key: cord-0280385-gp86ckf9
authors: Son, Hyeonwi; Zhang, Yan; Shannonhouse, John; Ishida, Hirotake; Gomez, Ruben; Akopian, Armen; Kim, Yu Shin
title: Mast cell-specific receptor/corticotropin-releasing factor axis regulates alcohol withdrawal-associated headache
date: 2021-05-22
journal: bioRxiv
DOI: 10.1101/2021.05.21.445199
sha: b8333975b5dac8f46802b82bb43f4df979cf0f6a
doc_id: 280385
cord_uid: gp86ckf9

Rehabilitation from alcohol addiction or abuse is challenging due to alcohol withdrawal symptoms. Headache is a severe alcohol withdrawal symptom that frequently contributes to rehabilitation failure. Despite the need for treating alcohol withdrawal-induced headache, there is no appropriate therapeutic option available. Development of improved therapeutics will depend on obtaining a clearer understanding of alcohol withdrawal-induced headache pain mechanisms. Here, we show that the mast cell-specific receptor MrgprB2 controls development of alcohol withdrawal-induced headache. Withdrawing alcohol from alcohol-acclimated mice induces strong headache behaviors, including facial allodynia, facial pain expressions, and reduced walking movement, symptoms often observed in humans suffering from headache. Observed pain behaviors were abolished in MrgprB2-deficient mice. We observed in vivo spontaneous activation and hypersensitization of trigeminal ganglia neurons in alcohol withdrawal mice, but not in MrgprB2-deficient mice. Corticotropin-releasing factor (CRF) was increased in dura mater after alcohol withdrawal. Injection of CRF into dura mater resulted in activation of trigeminal ganglia neurons and vasodilation, which was accompanied by headache behavior. In cells, CRF evoked Ca2+ transients via MrgprB2 or human MrgprX2. The results indicate that alcohol withdrawal causes headache via mast cell degranulation in dura mater. The process is under control of MrgprB2/MrgprX2, which would appear to represent a potential target for treating alcohol withdrawal-related headache.

Alcohol is an addictive substance, and approximately 380 million people suffer from alcohol abuse or dependence (WHO, 2016) . Global disasters, such as terrorism, economic adversity, and the COVID-19 respiratory epidemic, are associated with increased alcohol consumption and increased vulnerability to development of risky drinking behaviors 1 . Rehabilitation from alcoholism is of critical importance to managing alcohol dependency of a large portion of the population. However, the rehabilitation process is hindered by alcohol withdrawal symptoms, specifically, headache [2] [3] [4] . The temporary relief from alcohol withdrawal-induced headache pain derived from resumption of alcohol consumption is a driving force failure to break the addiction cycle 5, 6 , seriously affecting quality of life and aggravating alcohol dependence.

Despite a major unmet medical need for treating alcohol withdrawal-induced headaches, there is no appropriate therapeutic option available. To develop better therapeutics, it will be necessary to obtain a clearer understanding of alcohol withdrawal-induced headache pain mechanisms.

Headache is initiated from the activation of trigeminal ganglia (TG) neuronal afferents and vasodilation in dura mater. Processes that cause local inflammation of dura mater sensitize peripheral afferents of TG neurons [7] [8] [9] , and their activation by mechanical and chemical stimuli contribute to the progression of general headaches 7, 10 . Mast cell degranulation in dura mater has been implicated in local inflammation and nociceptive afferent activation of TG neurons and vasodilation 11, 12 , suggesting that activated mast cells in dura mater may mediate headache. Mast cells are located proximal to peripheral nerve endings and peripheral blood vessels in dura mater, where they can be activated by various secretagogues to release proinflammatory cytokines 13, 14 . Mas-related G-protein-coupled receptor B2 (MrgprB2), which is selectively expressed on connective tissue mast cells, is 4 activated by basic secretagogues 15 , and mediates neurogenic inflammation pain [16] [17] [18] . Chronic alcohol consumption induces increased mast cell numbers and degranulation in peripheral tissues, where mast cells could mediate inflammation [19] [20] [21] . In view of these observations, we tested whether mast cell-specific MrgprB2 contributes to alcohol withdrawal-induced headache.

In a two-bottle (10% ethanol vs. water) voluntary choice paradigm, mice exhibited a preference for ethanol (Extended Data Fig. 1a ). Neither food intake (Extended Data Fig. 1c) nor body weight (Extended Data Fig. 1d ) were affected. Ethanol preference increased gradually over the three week study (Extended Data Fig. 1b) 22 . However, the headache behaviors induced by alcohol withdrawal were abolished in MrgprB2-deficient (MrgprB2 KO) mice (Fig. 1a-c) . Thus, withdrawal from ethanol resulted in headache behaviors, and indicated that mast cell-specific MrgprB2 likely mediates alcohol withdrawal-induced headache.

Mast cell activation via MrgprB2 in dura mater involve in alcohol withdrawal-induced sensitization of TG neurons. To assess the activity of TG neurons in mice following ethanol 5 withdrawal after 3 weeks of alcohol access, we monitored the activity of TG neurons in intact live animals using in vivo TG Pirt-GCaMP3 Ca 2+ imaging. The total number (>141±6.34) of spontaneously activated neurons was dramatically increased in TG of alcohol withdrawal mice compared to water-fed controls ( Fig. 1d-f ). The spontaneously activated neurons included neurons exhibiting Ca 2+ oscillations and neurons with steady-state high Ca 2+ , both of which were significantly increased in alcohol withdrawal mice (Fig. 1d-f ). The group of small-diameter (<20 μm) and medium-diameter (20-25 μm) neurons from alcohol withdrawal mice showed more spontaneously activated neurons than water-fed controls ( Fig. 1g and Extended Data Fig. 1g ). The numbers of spontaneously activated TG neurons were significantly decreased in alcohol withdrawal of MrgprB2-deficient mice ( Fig. 1d-g) . Ethanol consumption is known to modulate mast cell activities, including increasing degranulation [19] [20] [21] . Indeed, degranulated mast cells and the total number of mast cells were increased in dura mater of ethanol drinking mice ( Fig. 1h and Extended Data Fig. 2a) , and these increases were abolished in MrgprB2-deficient mice ( Fig. 1h and Extended Data Fig. 2a) . These data indicate that MrgprB2 is required for mast cell degranulation evoked by alcohol withdrawal, suggesting that mast cell activation via MrgprB2 results in development of alcohol withdrawal-induced headache and pain behaviors.

Our findings indicate that degranulation of mast cells via MrgprB2 sensitizes TG neurons to evoke alcohol withdrawal-induced periorbital mechanical hypersensitivity and pain behaviors, but it is still unclear how alcohol withdrawal mediates activation of MrgprB2 leading to degranulation of mast cells. We sought to identify a putative activator of MrgprB2 after alcohol withdrawal.

Alcohol consumption and withdrawal is known to activate the hypothalamic-pituitary-adrenal (HPA) axis [23] [24] [25] [26] [27] . We postulated that Corticotropin-releasing factor (CRF), a hormone active in 6 regulating HPA axis function, might be involved in MrgprB2 activation and subsequent development of alcohol withdrawal-induced headache 28, 29 . We found significantly increased CRF localization and expression to the dura mater of alcohol withdrawal mice compared to water-fed controls ( Fig. 2a and Extended Data Fig. 2b ). Since CRF, a secretagogue of mast cells, induces mast cell degranulation 30 , we hypothesized that CRF-induced activation of mast cells mediates alcohol withdrawal-induced headache behaviors. To confirm whether local increases of CRF in dura mater induce headache-like and pain behaviors, CRF was directly injected into dura mater. CRF injection significantly induced periorbital mechanical hypersensitivity from 1 to 24 hrs post-injection, which returned to baseline within 72 hrs (Fig.   2b ). This was similar to the effect of IL-6, which is a well-known periorbital mechanical hypersensitivity inducer 31, 32 . However, MrgprB2-deficient mice did not show periorbital hypersensitivity following dural CRF injection (Fig. 2b) . Recent studies reported that MrgprB2 responds to various positively charged protein-secretagogues 15, 33 , which led us to test whether CRF directly activates MrgprB2. We confirmed that CRF induced Ca 2+ transients in HEK293 cells expressing MrgprB2 or the human ortholog MrgprX2 (Fig. 2c,d) .

We also applied CRF onto isolated mouse mast cells and cultured LAD2 human mast cells in vitro, and found that the CRF application also evoked Ca 2+ transients in isolated mouse mast cells and LAD2 human mast cells (Fig. 2e ,f). To rule out the possibility that CRF indirectly activated MrgprB2 in mast cells by engaging CRF-induced Ca 2+ transients via CRF receptors, we added astressin, a CRF 1/2 receptor inhibitor, before CRF application. Astressin treatment did not block CRF-induced Ca 2+ transients in isolated mouse mast cells or LAD2 human mast cells but did increase Ca 2+ transients (Fig. 2g,h) . Consistent with this result, astressin injection into dura mater enhanced the effect of CRF on periorbital mechanical hypersensitivity (Extended Data Fig. 2c ). In addition, using in vivo TG Pirt-GCaMP3 Ca 2+ 7 imaging, we found a significant increase in the number of activated neurons following dural CRF injection (Fig. 2i) . In in vivo dura mater imaging, direct CRF application onto dura mater induced vasodilation (Fig. 2j) , a primary event occurring during migraine and headaches 7, 34 . These results indicate that CRF, which is likely released from dural blood vessels, induces mast cell degranulation via MrgprB2 activation, and regulates development of alcohol withdrawal-induced headache and pain behaviors by sensitization of TG nerve in dura mater.

From studies identifying the pathophysiology of migraine headache, it is known that cutaneous allodynia is observed by non-noxious stimuli to periorbital and forehead skin areas during headache 35 . To confirm sensitization of TG nerve with various stimuli, including mechanical, thermal, and chemical stimuli, we applied von Frey filament (mechanical), hot water (thermal), or capsaicin (chemical) to each orofacial region innervated by ophthalmic (V1), maxillary (V2), or mandibular (V3) branches of TG nerves during in vivo TG Pirt-GCaMP3 Ca 2+ imaging. Application of 0.4 g to the V1 region, but not to V2 or V3 regions, revealed increased activation of TG neurons in alcohol withdrawal mice ( Fig. 3b and Extended Data Fig. 3a ,h). This signal was due to increased activation of small-diameter to medium-diameter neurons (Fig. 3b ). Mild hot water (40°C) or acetone (cold stimulus) applications also revealed greater numbers of activated TG neurons in V1 region but not in V2 or V3 regions ( Fig. 3c and Extended Data Fig. 3C ,J,E,L). The number of small to medium-diameter neurons activated in V1 region was increased in response to mild hot water (40°C) in alcohol withdrawal mice (Fig. 3c) . Capsaicin injection into the V1 region but not in V2 or V3 regions of alcohol withdrawal mice resulted in an increase in activated TG neurons ( Fig. 3d and Extended Data Fig. 3g,n) . The sensitization of TG neurons in V1 region were also observed 8 in alcohol withdrawal mice withdrawn from voluntary ethanol consumption after as long as 8 weeks (Extended Data Fig. 3p,s) . Consistent with the results of headache and pain behaviors, MrgprB2-deficient mice after alcohol withdrawal did not exhibit an increase in activated TG neurons relative to control groups in response to all stimuli in all three branches (Fig. 3b-d) .

Collectively, these results suggest that alcohol withdrawal mice exhibit sensitization of TG nerve by mechanical, thermal, and chemical stimuli, and suggest that the mast cell-specific receptor MrgprB2 contributes to alcohol withdrawal-induced sensitization of TG nerves responsible for inducing headache and pain behaviors. imaging. Mild (100 g) or noxious (300 g) press to the hindpaw of alcohol withdrawal mice significantly increased the number of activated DRG neurons compared to water-fed controls ( Fig. 4c-h) . In response to these stimuli, small-and medium-diameter neurons were significantly more activated in DRG of alcohol withdrawal mice than water-fed controls ( Fig.   4e ,h). We also found an increase in activated neurons in the DRG of alcohol withdrawal mice in response to noxious heat (50°C) ( Fig. 4i-k) . The numbers of small-and medium-diameter neurons activated by noxious heat were significantly higher in alcohol withdrawal mice than in water-fed controls (Fig. 4k) .

To identify specific molecular signalling mechanisms that drive hypersensitivity in different anatomical locations as a result of alcohol withdrawal, we investigated whether inhibitor of tumour necrosis factor-α (TNF-α) receptor, which was increased by alcohol consumption 39 , would block alcohol withdrawal-induced mechanical allodynia. We found that alcohol withdrawal-induced mechanical allodynia in hindpaw or in the head was blocked by R-7050, a TNF-α receptor inhibitor (i.p. injection for 11 consecutive days) (Fig. 4l,m) . A recent study suggested that chronic alcohol consumption induces nociceptor sensitization in hindpaw through an increase in reactive oxygen species (ROS) 37 , which led us to ask whether ROS production is a consequence of TNF-α release, and if ROS are involved in both hindpaw and head mechanical allodynia. We found that mechanical allodynia in hindpaw of alcohol withdrawal mice was reversed by PBN, a ROS scavenger, whereas mechanical allodynia in the head was not reversed (Fig. 4n,o) . We also confirmed that the mechanical allodynia in the head caused by alcohol withdrawal was reversed by SB366791, a TRPV1 inhibitor (Fig.   4n ,o). Activated mast cells can release TNF-α, which is a well-known potentiator of TRPV1 ion channel 40 , suggesting that in alcohol withdrawal mice TRPV1 activation is a component of a downstream signalling cascade of mast cell activation via MrgprB2. These mechanisms are independent pathways from CRF receptor signaling.

Clinical observations suggested that alcohol drinking causes headaches [41] [42] [43] [44] , but the pathophysiological mechanism still remains unknown. Here, we show that withdrawal from alcohol drinking causes headache and pain behaviors, which are associated with sensitization of TG neurons. We also show that CRF directly activates MrgprB2, which mediates alcohol withdrawal-induced behavioral and cellular changes (Fig. 4p ). Moreover, we identified different signaling mechanisms of alcohol withdrawal-induced hypersensitivity in the head 1 0 compared with hypersensitivity in hindpaw. These results identify a mechanism of alcohol withdrawal-induced headache, and point to a therapeutic target for treating alcohol withdrawal-induced headache problems and addictive behaviors.

Mast cells in dura mater participate in inflammation and then sensitization of peripheral afferents, which are considered as a cascade of the development of migraine headache 12, 45 . We here demonstrate that CRF activates mast cells via Mrgprb2, which causes headache behaviors and the TG nerve sensitization. These results indicate that CRF mediates alcohol withdrawal-induced physiological changes and mast cell degranulation that produces headache. Since alcohol withdrawal can induce changes in various hormones or peptides, there is a possibility that upregulation or downregulation of other hormones or peptides can affect alcohol withdrawal-induced behavioral and cellular changes via mast cell activation.

Mast cells express CRF1/2 receptors. Unlike Mrgprb2, CRF receptors act as modulators and/or indirectly affect mast cell activity. It has been reported that CRF1 receptor enhances calcium transient and calcium signal and CRF2 receptor suppresses it in mast cells 46 . We here confirm that the inhibition of CRF1/2 receptors enhances CRF-induced mast cell activation.

Because expression of CRF receptors is changed under various conditions 46 , further study is needed about the modulatory role of CRF receptors in mast cells of alcohol withdrawn mice.

The withdrawal-induced physiological changes such as CRF elevation also occur in the use of other addictive substances, including heroin and other opioids 47 . Thus, CRF-MrgprB2 axis-induced mast cell activation might be a significant signaling pathway in the development of headaches induced by addictive substances.

Pain and alcohol dependence have a close relationship. Acute pain-inhibitory effect by alcohol consumption and withdrawal from alcohol drive to motivate alcohol drinking, 1 1 use disorder exhibit more significant pain responses during the early stages of alcohol abstinence. In adults with chronic pain, their pain intensity is correlated with increased alcohol consumption 3 . Therefore, pain relief, especially from headaches could be a way to lower severe alcohol dependence 5 .

Alcohol abuse or addiction is a significant public health problem, especially during the COVID-19 pandemic because the pandemic is leading people to drink alcohol a lot more than before. Indeed, the pandemic increases vulnerability to the development of alcohol addictive behaviors 1 . Moreover, pains such as headaches from alcohol withdrawal disrupt rehabilitation from alcohol addiction and/or abuse 3, 5 . We here demonstrate that the blockade of mast cell-specific MrgprB2 in mast cells attenuates alcohol withdrawal-induced headache behaviors. Therefore, our results provide CRF/MrgprB2 as new therapeutic targets for treating alcohol withdrawal-induced headaches and even for alcohol addiction and/or abuse. 

Frey test was performed according to previously published methods 32, 36 . Mice were familiarized and habituated to the experimenter's smell, hand touch, and eye contact for at least for 3 days, and then were acclimated in a plexiglass chamber with 4 oz paper cups for 2 h/d for 3 days. After acclimation, mice were subjected to baseline testing of cutaneous facial = clearly present) as previously published 51 . The open field test was performed in a new cage for 5 min. The mouse's movement was recorded using a video camera, and the movement was analysed using ImageJ (NIH) with animal tracker (plugin) 52 .

imaging. DRG exposure surgery was performed as previously described 48 DRG transverse process was removed to expose underlying DRG. Bleeding from the bone was stopped using styptic cotton. For TG exposure surgery, we first surgically exposed the right side dorsolateral skull by removing skin and muscle. The dorsolateral skull (parietal bone between right eye and ear) was removed using a dental drill (Buffalo Dental

Manufacturing, Syosset, NY, USA) to make a cranial window hole (~10X10 mm). The TG was then exposed where it is located under the brain by aspirating overlying cortical tissue through a cranial window in the dorsolateral skull. The animal was then laid on its abdomen on the stage under a Zeiss LSM 800 confocal microscope (Carl Zeiss). The animal was restrained using a mouse tooth holder to minimize movements from breathing and heartbeats.

During the surgery, the body temperature of the mouse was maintained on a heating pad at 37°C ± 0.5°C as monitored by rectal probe. 1 9 In vivo Pirt-GCaMP3 Ca 2+ imaging in DRG and TG. In vivo Pirt-GCaMP3 Ca 2+ imaging in live mice was performed for 1-5 hours immediately after exposure surgery as previously described 48, 49 . After the exposure surgery, mice were laid abdomen-down on a custom-designed platform under the microscope objective. For in vivo DRG Pirt-GCaMP3

Ca 2+ imaging, the spinal column was stabilized using two clamps on vertebra bone above and below the DRG being imaged. Live images were acquired at ten frames per cycle in frame-scan mode per ~4.5 to 8.79 s/frame, at ranging from 0 to 90 μm, using a 10 X 0.4 NA dry objective at 512 X 512 pixel or higher resolution with solid diode lasers tuned at 488 nm wavelength and emission at 500-550 nm. An average of 1,825±71 neurons per DRG (~10-15% of total DRG neurons) was imaged. For in vivo TG Pirt-GCaMP3 Ca 2+ imaging, the animal's head was fixed by a custom-designed head holder. During the imaging session, body temperature was maintained at 37°C ± 0.5°C on a heating pad and monitored by rectal probe.

Anaesthesia was maintained with 1-2% isoflurane using a gas vaporizer, and pure oxygen was delivered through a nosecone. Live images were acquired at ten frames per cycle in frame-scan mode per ~4.5 to 8.79 s/frame, at ranging from 0 to 90 μm, using a 5 X 0.25 NA dry objective at 512 X 512 pixel or higher resolution with solid diode lasers tuned at 488 nm wavelength and emission at 500-550 nm. An average of 2,867±87 neurons per TG (~10% of total TG neurons 53, 54 ) was imaged and small regions of TG neurons were imaged at faster speed >40Hz. von Frey filaments (0.4 g, and 2.0 g) were applied to the face or hindpaw of exposed TG branches or DRG side. 100 g and 300 g press were applied to the whole palm of hindpaw using rodent pincher (Bioseb, U.S.A.). Whole hindpaw or different TG branches at animal's face was applied by 40 °C, 50 °C, or 60 °C water and acetone was applied by pipette to the hindpaw or the different TG branches. Capsaicin (500 μM, 10 μ l) or KCl (500 mM, 10 2 0 μ l) was cutaneously injected into the different TG branches using a 0.5-ml insulin syringe with a 28-gauge needle.

In vivo Pirt-GCaMP3 Ca 2+ imaging data analysis. For imaging data analysis, raw image stacks were collected, deconvoluted, and imported into ImageJ (NIH). Optical planes from sequential time points were re-aligned and motion corrected using the stackreg rigidbody cross-correlation-based image alignment plugin. Ca 2+ signal amplitudes were expressed as F t /F 0 as a function of time. 

Mouse dura injection was performed as previously described 31 .

Mice were anesthetized under isoflurane briefly, and drugs or compounds were injected in a volume of 5 µl via a modified internal cannula (P1 Technologies, Roanoke, VA, USA). The length of injection needle was adjusted to 0.5 to 0.65 mm.

In vivo blood vessel imaging in dura mater. Mice conjugated 2k Dalton dextran (Nanocs) was injected into the tail vein (100 μl of 1 mg/ml in saline) to visualize blood vessels in dura mater. Anesthetized mice were imaged with a single photon confocal microscope (Carl Zeiss) using the 40X water immersion with 1.0 NA objective. The vessel diameter was measured using ImageJ software.

Immunofluorescence imaging in dura mater. Animals Zeiss Examiner/A1 microscope fitted with a 40× water-immersion objective (0.75-NA, 2.1mm free working distance, Carl Zeiss) and with an Axiocam 705 color camera (Carl Zeiss).

Fluorescence images were taken alternately every 5 s using the Zeiss Zen Blue software module. Cells were imaged in SES at room temperature; drugs were bath applied into the chamber following 10 cycles of baseline imaging, and responses were monitored for an additional 50 cycles. The percentage of cells responding to the CRF among total tdTomatoexpressing cells was calculated to quantify the CRF response.

Western blot for CRF of dura mater. Tissue lysates were prepared and were analyzed with western blot as previously described 55, 56 . Briefly, the collected dura mater was homogenized and centrifuged for 30 min at 12,000 rpm. The protein samples were separated by SDS-PAGE and then transferred to a PVDF membrane (Amersham, Buckinghamshire, UK), which was immunoblotted with anti-CRF (Santa Cruz, 1:500) and anti-β-actin (Cell Signaling, 1:5000) 57 . Each expression was quantitated using ImageJ (NIH). Protein expression was normalized to β -actin in the same sample.

Statistical analysis. Group data were expressed as mean ± S.E.M. Two-tailed unpaired Student's t test, one-way ANOVA tests, and two-way ANOVA tests were used to determine significance in statistical comparisons, and differences were considered significant at p < 0.05. 

Alcohol Use and COVID-19: Can we Predict the Impact of the Pandemic on Alcohol Use Based on the Previous Crises in the 21st Century?

Alcohol dependence as a chronic pain disorder

Interrelations between pain and alcohol: An integrative review

Clinical characterization of delayed alcohol-induced headache

Drug Addiction : Hyperkatifeia / Negative Reinforcement as a Framework for Medications Development

Chronic inflammatory pain drives alcohol drinking in a sex-dependent manner for C57BL/6J mice

A Systems Neuroscience Approach to Migraine

CGRP and the Trigeminal System in Migraine

Human models of migraineshort-Term pain for long-Term gain

Sensitization of meningeal sensory neurons and the origin of headaches

Shared fate of meningeal mast cells and sensory neurons in migraine

Migraine pain, meningeal inflammation, and mast cells

Mast cell/nerve interactions in vitro and in vivo

Different activation signals induce distinct mast cell degranulation strategies

Identification of a mast-cell-specific receptor crucial for pseudoallergic drug reactions

Getting It Wrong and Right Simultaneously

Cellular and Molecular Mechanisms of Pain

A Role for Mast Cells in Alcohol-Induced Tissue Damage and Remodeling

Ethanol-Induced Mast Cell-Mediated Inflammation Leads to Increased Susceptibility of Intestinal Tumorigenesis in the APCΔ468 Min Mouse Model of Colon Cancer

Mast cells and ethanol consumption: Interactions in the prostate, epididymis and testis of UChB rats

Update on Animal Models of Migraine

Effect of chronic alcohol consumption on the activity of the hypothalamic-pituitary-adrenal axis and pituitary β -endorphin as a function of alcohol intake, age, and gender

The effects of alcoholism on the hypothalamic-pituitary adrenal axis: Interaction with endogenous opioid peptides

The effects of mild ethanol intoxication on the hypothalamic-pituitary-adrenal axis in nonalcoholic men

Developmental expression of glial fibrillary acidic protein and glutamine synthetase mRNAs in normal and jimpy mice

Activation of Brain NOP Receptors Attenuates Acute and Protracted Alcohol Withdrawal Symptoms in the Rat

Mast cells in stress, pain, blood-brain barrier, neuroinflammation and alzheimer's disease. Front

The role of mast cells in migraine pathophysiology

Corticotropin-releasing hormone induces skin mast cell degranulation and increased vascular permeability, a possible explanation for its proinflammatory effects

Non-invasive dural stimulation in mice: A novel preclinical model of migraine

Dural calcitonin gene-related peptide produces female-specific responses in rodent migraine models

Recent advances in our understanding of mast cell activation -or should it be mast cell mediator disorders?

Vascular contributions to migraine: Time to revisit? Front

An association between migraine and cutaneous allodynia

Anterior cingulate cortex contributes to alcohol withdrawal-induced and socially transferred hyperalgesia

Schwann cells expressing nociceptive channel TRPA1 orchestrate ethanol-evoked neuropathic pain in mice

Key Role for the Epsilon Isoform of Protein Kinase C in Painful Alcoholic Neuropathy in the Rat

New Implications for the Melanocortin System in Alcohol Drinking Behavior in Adolescents : The Glial Dysfunction Hypothesis

Targeted overexpression of tumor necrosis factor-α increases cyclindependent kinase 5 activity and TRPV1-dependent Ca2+ influx in trigeminal neurons

Prospective cohort study of daily alcoholic beverage intake as a potential trigger of headaches among adults with episodic migraine

Alcohol-induced headaches: Evidence for a central mechanism?

The alcohol hangover

Hangover headache: Accompanying symptoms. Vågå study of headache epidemiology

Mast cell degranulation activates a pain pathway underlying migraine headache

Mast cell corticotropin-releasing factor subtype 2 suppresses mast cell degranulation and limits the severity of anaphylaxis and stress-induced intestinal permeability

Drug withdrawal conceptualized as a stressor

Coupled Activation of Primary Sensory Neurons Contributes to Chronic Pain

Central terminal sensitization of TRPV1 by descending serotonergic facilitation modulates chronic pain

Quantitative assessment of tactile allodynia in the rat paw

Coding of facial expressions of pain in the laboratory mouse

AnimalTracker: An ImageJ-Based Tracking API to Create a Customized Behaviour Analyser Program

Requirement of neurotrophin-3 for the survival of proliferating trigeminal ganglion progenitor cells

Primary sensory neuron addition in the adult rat trigeminal ganglion: Evidence for neural crest glio-neuronal precursor maturation

Bioactive forms of vitamin D selectively stimulate the skin analog of the hypothalamus-pituitary-adrenal axis in human epidermal keratinocytes

Chronic exposure to glucocorticoids induces suboptimal decisionmaking in mice

Detecting multiple proteins by Western blotting using same-species primary antibodies, precomplexed serum, and hydrogen peroxide

We thank Gregory Dussor and members of the Gregory Dussor laboratory for advice on the facial von Frey test and dura injection. This work was supported by National Institutes of 

EtOH Water

The authors declare no competing interests.