Keywords: Aryl hydrocarbon receptor nuclear Cortical spreading depression c-Src: Cellular-sarcoma CTD: Chronic tryptophan depletion Cytochrome P450 family 1 subfamily Familial hemiplegic migraine GABA: γ-Aminobutyric acid GFAP: Glial fibrillary acidic protein Indole-3-acetaldehyde IAld: Indole-3-aldehyde Inflammatory bowel disease IBS: Irritable bowel syndrome ICHD-3: International classification International headache society Lamina II inner Lamina II outer IL: Interleukin ILA : Indole-3-lactic acid IPA : Indole-3-propionic acid IPYA: Indole-3-pyruvic acid ISDN: Isosorbide dinitrate ITE: 2-(1′H-indole-3′-carbonyl)thiazole- Mitogen-activated protein kinase Paraventricular hypothalamic nucleus Spinal trigeminal nucleus caudalis Signal transducer and activator Trigeminocervical complex TCDD: 2, 3, 7, 8-tetrachlorodibenzo-pdioxin Transient receptor potential ankyrin Ventral posteromedial nucleus XAP2: X-associated protein 2 XRE: Xenobiotic response element

Migraine is a neurological disease with an episodic nature. In 3% of patients, migraine progresses over time to reach a chronic state. Although the mechanisms underlying this chronification remain unknown, we hypothesize that a generalized inflammatory state coupled with impairment of tryptophan (TRP) metabolism could be a risk factor. TRP-derived metabolites are involved in neuronal modulation, regulate inflammation by activating the aryl hydrocarbon receptors (AhR), and play a pivotal role in migraine pathophysiology. Here, using biomolecular, immunohistochemistry, and behavioral approaches in a mouse model of migraine induced by systemic isosorbide dinitrate (ISDN) administration, we assessed the contribution of the TRP metabolism on the inflammatory responses during migraine progression. Two migrainerelated regions were focused on, the noradrenergic locus coeruleus (LC) nucleus, and the spinal trigeminal nucleus caudalis (Sp5C). A single ISDN injection evoked cutaneous and light hypersensitivities, two prominent migraine-related symptoms, which were linked to enhanced systemic pro-inflammatory cytokine release. Repeated ISDN administrations also induced astrogliosis in the LC. Chronic TRP deficiency promoted microglial activation and noradrenergic dysfunction in the LC and worsened the intensity and duration of ISDN-induced cutaneous hypersensitivity in female mice.

Interestingly, this process was concomitant with increased inflammation-related indoleamine 2,3-dioxygenase (IDO) activity and pro-nociceptive AhR action. TRP deficiency also blocked the efficacy of sumatriptan (an anti-migraine drug) in both male and female mice, coupled with an upregulation of the expression of subtype 1D of the serotoninergic receptors in the Sp5C. This project shows that migraine progression is associated with a systemic and local inflammation promoted by impaired TRP metabolism, which alters the descending pain modulatory system, and the abortive therapeutic efficacy of triptans.

ACKNOWLEDGMENTS

First of all, I would like to thank all the members of the jury for their time and effort. Thank you, Pr. Marc Landry, Pr. Dieudonnée Togbe, and Dr. Cyril Rivat for agreeing to be reporters of my work. Thank you, Pr. Christine Courteix and Dr. Michel Lanteri-Minet for agreeing to be my thesis examiners.

To my thesis supervisor, Dr. Cristina Alba-Delgado. I remember clearly the first time I ever stepped foot in France and you were the one picking me up from the airport. From day one you were not only my supervisor but my friend. Cristina, I can't even begin to thank you for all the support, attentiveness, patience, and confidence you have given me. I consider myself lucky to have had the chance to go through this thesis with a supervisor like you and to be able to learn all that I have within these three years.

Words can't express my gratitude to you for all your input that made this endeavor possible. Thank you for always being there with your optimism and ambition to answer my questions and discuss with me the ins and outs of our project. And also to discuss plans, food, experiences, and everything else. Forever a Tryptophan Team Member! I would like to sincerely thank my second thesis supervisor, Pr. Radhouane Dallel. Professor, you have given me the opportunity to be a part of your lab and I am forever thankful for that. Thank you for being there to listen when I had questions and for all your great input into this project.

Isabelle, thank you for all the great advice and help you have given me through this journey. You were always someone who I can come to when I needed help. Thank you for always being there with your gentle ways and full of optimism.

François thank you for all the help in my project and the time you put into my data, but not only that thank you for all the conversations about France and learning more about this country and for all the advice and support. And thank you for the amazing game nights with the lab! Karine, your sweet heart and care were always there. I can't begin to thank you for all your help. And of course a huge heart every time I see you! Anne Marie & Amelie. Anne Marie, the adoptive mother of all of us. I am forever thankful for all your morning hugs and your affection. Amelie always with a smile, thank you for being there at times when I was feeling low. Plein des bisous!! Sylviane, you have helped me a lot not only with the animals but also with listening and being a good friend. Thank you for all that! Gisela, every time I was running around in the histology room I would always search for you for your input. You were always a great help to me. Thank you for being there and giving me great advice for the future. Next time I visit Porto I will for sure tell you! Christelle and Anne. I can't imagine how many times we talked about teeth and dentists! Thank you for being so supportive, gentle, kind, and caring. Lénaïc, Cedric, Myriam, Ahmad, Michael, and Philippe, thank you for the great working experience with all of you. It's been a real pleasure to know and work with you.

To all the students that I have helped mentor and who have added so much effort and input to this thesis. Anaïs, Vido, Manon, Robin, Tamara, Marion, Stephanie, Guillaume, Rayan, and Ilias. You guys have all been amazing and I am very thankful to have had the opportunity to teach you and learn from you. And for all the moments of laughter and joy, we shared, I am very thankful.

Ornella, thank you for being a friend and for all the moments we shared. I hope to always have reunions whenever I visit France! Jenny, you were the first person I ever met when I came here, who helped me through every single step and was not only a friend but an older sister. I am so thankful for you Jenny! To my girls! Manon, my petite Olaf, and stitch twin. I can't even begin to say how thankful I am to have met you during these couple of years. I am so happy to take with me a lot of our Disney memories and random moments. So much love for you monocle!

The trio, my jeanouu, and pawpaw. I can't even begin to express how much I am going to miss you two! After spending three years in the same office together I for sure got used to our routine and our random shenanigans. I am so thankful for the huge bag of memories I will be carrying with me back home. From our outings, inside jokes, Channing Tatum, to Britney Spears. We have been through so much together both positive and negative but no matter what we were always each other's support. You both have made this experience a thousand times brighter and I can't and don't want to imagine doing this whole thing without you. I love you, my girls! Zazu and Hamada, to the two people that I call my best friends. Even with one of you being in Lebanon and the other in Germany, you both were a huge support system for me. I can't wait to celebrate more milestones with you by my side even from miles away! To the people that matter the most my parents, brother, and fiancé. Kas, my love, my fiancé, my baby, and my hubby-to-be. Even with the distance separating us and us being in two different continents and with our 10 hour time difference you always knew how to find ways to remind me that you are my support.

Words will never be enough to explain how thankful I am for you and how much I love you. Thank you for always being my support and holding my hand through it all. I can't wait to spend the rest of our lives together and to share many more milestones.

Hopefully our next step in Australia will be full of happiness and success. I love you my bubby!

To my Jado, thank you for not only being my brother and my support but my rock at times when I didn't know what to do. Thank you for always having my back and reminding me that all will be great in the end. To sharing many more successful moments. I love you more than anything my heart.

Mama and Papu, you are my biggest supporters. All the words of thanks and love that I have in my heart can't be written on paper. But if I had to say it in simple words, I wouldn't be the person that I am today without you. Thank you for being my rock when things got hard. For always pushing me to be the best I can be and to never give up on my dreams. Thank you for raising me to always dream and aim high, and that the sky is the limit. Packing my bags and leaving for this adventure wasn't easy at all but we know it's worth this moment right here. Your prayers and love are what brought me here and helped me to never give up on this dream that now became a reality. My love for you is endless. I love you!! To the little girl who never gave up on her dreams , you did it ! Be proud of yourself for you are now a doctor and all this was the fruit of your hard work and perseverance.

RÉSUMÉ

La migraine est une maladie neurologique de caractère épisodique. Chez 3% des patients, elle progresse avec le temps vers un état chronique. Bien que les mécanismes sous-jacents à cette chronification restent inconnus, nous émettons l'hypothèse qu'un état inflammatoire généralisé, couplé à une dérégulation du métabolisme du tryptophane (TRP), pourrait être un facteur de risque. Les dérivés du TRP sont impliqués dans la modulation neuronale, régulent l'inflammation en activant les récepteurs d'aryl hydrocarbone (AhR), et jouent un rôle central dans la physiopathologie de la migraine.

Ici, en utilisant des approches biomoléculaires, immunohistochimiques et comportementales dans un modèle murin de migraine induite par l'administration systémique de dinitrate d'isosorbide (ISDN), nous avons évalué la contribution du métabolisme TRP à la réponse inflammatoire au cours de la progression de la migraine.

Deux régions liées à la migraine ont été ciblées, le locus coeruleus (LC) noradrénergique et le noyau caudal du trijumeau (Sp5C). L'injection aigüe de l'ISDN a provoqué une hypersensibilité cutanée et à la lumière, deux des symptômes migraineux les plus rependus, en parallèle d'une augmentation de la libération systémique de cytokines pro-inflammatoires. L'administration répétée de l'ISDN a induit en outre une astrogliose dans le LC. La déficience chronique en TRP a favorisé l'activation microgliale et déréglé la fonction noradrénergique dans le LC, et aggravé l'intensité et la durée de l'hypersensibilité cutanée induite par l'ISDN chez les souris femelles. Fait intéressant, ce processus était concomitant avec une activité accrue de l'indoleamine 2,3-dioxygénase (IDO) liée à l'inflammation et une action pro-nociceptive des AhR. La déficience en TRP a également bloqué l'efficacité du sumatriptan (un médicament antimigraineux) chez les souris mâles et femelles, couplée à une augmentation de l'expression du sous-type 1D des récepteurs sérotoninergiques dans le Sp5C. Ce projet montre que la progression de la migraine est associée à une inflammation systémique et locale favorisée par une altération du métabolisme du TRP, qui altère l'activité des contrôles descendants de la douleur, et l'efficacité thérapeutique des triptans.

LIST OF FIGURES

PREFACE

This thesis is comprised of multiple sections, starting with an analysis of the background (Introduction), the hypotheses and objectives, the main results which are written in the format of a "manuscript to be sent for publication", and a section with complementary results. Finally, it concludes with a general discussion of the main results and conclusions.

Moreover, this work was presented in multiple national and international conferences :

Local Contributions

From our Neuro-Dol Laboratory, Team II:

• Dr. François Gabrielli and Mrs. Karine Herault

. Definition, Epidemiology, and Diagnosis

Migraine is a frequent incapacitating neurobiological disorder characterized by severe, unilateral, pulsating headache attacks, often accompanied by nausea and/or vomiting as well as sensory and motor disturbances (IHS 2018). A recent epidemiological study estimated the global prevalence of migraine at 14-15%, ranked as the third most common disease in the world in both males and females [START_REF] Steiner | Global Epidemiology of Migraine and Its Implications for Public Health and Health Policy[END_REF]. Although migraine affects 3 to 10% of children [START_REF] Borsook | Sex and the Migraine Brain[END_REF], incidence peaks between the ages of 20-24 years in women and 15-19 years in men, with 50% of first attacks occurring before the age of 25 years and in 75% before 35 years of age [START_REF] Stewart | Cumulative Lifetime Migraine Incidence in Women and Men[END_REF]. Worldwide prevalence data from the 2015 Global Burden of Disease Study show that migraine is two to three times more prevalent in women than in men between 30 and 50 years of age; after 50 years of age, the prevalence declines to ~5% in both men and women [START_REF] Uthman | Global, Regional, and National Disability-adjusted Life Years (DALYs) for 315 Diseases and Injuries and Healthy Life Expectancy (HALE) for 195 Countries and Territories, 1990-2015 : A Systematic Analysis for the Global Burden of Diseases, Injuries, and Risk Factors (GBD) 2015 Study[END_REF].

According to criteria defined by the International Classification of Headache Disorders (ICHD-3) established by the Headache Classification Committee of the International Headache Society (IHS), the two most common types of migraine are migraine without aura (formerly known as common or simple migraine), which affects approximately 70% of people (mainly women during reproductive years); and migraine with aura, in approximately 20% of cases. The remaining 10% of people suffer from both types of attacks [START_REF] Macgregor | Diagnosing Migraine[END_REF]. Migraine diagnosis is based on a set of guidelines, summarized in Table (1). Migraine without aura is characterized by headache attacks lasting 4-72 h (when untreated or unsuccessfully treated), unilateral with a pulsating quality ranging from moderate to severe in intensity, aggravated by routine physical activity, and associated with nausea, and/or photophobia (hypersensitivity to light) and/or phonophobia (hypersensitivity to sound). Migraine with aura encompasses a transitory-focused neurological symptom, termed aura, mostly consisting of visual disturbances but sometimes also concerning motor or speech impairments that frequently precede or occasionally coincide with the headache. Its prevalence increases with age, occurring in around 13% of attacks in migraineurs aged 18-29 years, 20.1% between 40-49 years, and 41% by age 70 years or older. Only 1% of patient experience migraine with aura without headache (Bigal and Lipton 2006). Although migraine is not life-threatening in itself, the severity and frequency of attacks can result in significant disability and reduced quality of life, even between attacks. According to the Global Burden of Disease Study migraine on its own is the sixth leading cause of disability-adjusted life years (DALY) score in the age group 25-39 years, tenth in Western Europe [START_REF] Uthman | Global, Regional, and National Disability-adjusted Life Years (DALYs) for 315 Diseases and Injuries and Healthy Life Expectancy (HALE) for 195 Countries and Territories, 1990-2015 : A Systematic Analysis for the Global Burden of Diseases, Injuries, and Risk Factors (GBD) 2015 Study[END_REF]. Despite acute and/or prophylactic medications, 90% of patients report a negative impact of migraine on their overall quality of life [START_REF] Estave | Learning the Full Impact of Migraine through Patient Voices: A Qualitative Study[END_REF]), which limits their capacity for social and professional activities. Migraine controls one's life and/or makes life difficult. The fear of migraine attacks often creates anticipatory behaviors, amplifying migraine's effects with increased anxiety, pain catastrophizing, isolation, and hopelessness. Difficulty concentrating, communicating, and experiencing irritability are disabling ictal symptoms that affect functionality beyond pain and the typical associated migraine symptoms (e.g. photophobia, phonophobia, nausea, and vomiting). Migraine is also an expensive illness: in Europe, the mean per-person annual cost of migraine is estimated to be €1222 (M. [START_REF] Linde | The Cost of Headache Disorders in Europe: The Eurolight Project[END_REF]. A third of the financial burden is linked to the health care system (including outpatient and inpatient care, prescriptions, and emergency care), while two-thirds is linked to indirect costs associated with absenteeism (full days of productive workforce loss) and/or presenteeism (days worked with diminished productivity) [START_REF] Edmeads | The Economic Impact of Migraine: An Analysis of Direct and Indirect Costs[END_REF]. No overall data for differences between men and women have been reported.

Clinical Phases of a Migraine Attack

Migraine is described as having distinct and discrete phases: premonitory, aura (in the case of migraine with aura), headache, and post-headache (Figure 1). In migraineurs, the median duration of an untreated headache attack is one day but can range from 4 h (2 h in children) to 3 days [START_REF] Jensen | Epidemiology and Comorbidity of Headache[END_REF]. The duration is longer if premonitory and postdromal symptoms are included. The premonitory phase, also termed the prodromal phase, affects 77% of patients, being more prevalent in females (81%) than males (64%) [START_REF] Laurell | Premonitory Symptoms in Migraine: A Cross-Sectional Study in 2714 Persons[END_REF].

This phase starts 24 to 48 h before the headache and is associated with a plethora of symptoms, including fatigue, neck stiffness, mood and cognitive changes, photophobia, phonophobia, and osmophobia (hypersensitivity to odors). Other less frequent symptoms are yawning and food cravings [START_REF] Eigenbrodt | Premonitory Symptoms in Migraine: A Systematic Review and Meta-Analysis of Observational Studies Reporting Prevalence or Relative Frequency[END_REF].

The aura phase occurs in over 90% of migraineurs suffering from migraine with aura (IHS 2018), and can precede the attack or be present simultaneously. The aura is typically experienced as visual symptoms characterized by reversible manifestations (such as flashes of light, blind spots, or visualizing 'zig zag' patterns), starting near the center of vision and spreading peripherally over 20-30 min. Sensory aura, such as pins and needles or numbness spreading up one arm into one side of the face and mouth, and transient speech disturbances, such as trouble in understanding words and/or wordfinding difficulty, are less prevalent and, if present, are almost always accompanied by visual aura (IHS 2018).

The headache phase is characterized by moderate to severe frequently unilateral pulsatile, throbbing pain with an augmenting intensity within the first few hours lasting from 4 to 72 h in duration, without adequate treatment (IHS 2018). The throbbing aspect is often coupled with nausea, vomiting, photophobia, and/or phonophobia.

Patients also report cutaneous hypersensitivity, also called cutaneous allodynia, which is defined as pain due to a non-noxious stimulus. Aggravation of the headache could occur due to mild physical activity hence patients may seek relief in dark places and require bedrest during the attack.

The postdrome phase encompasses persistent symptoms after headache cessation. The symptoms reflect those experienced during the premonitory stage, such as exhaustion, tiredness, difficulty concentrating, and neck stiffness (Nicola J. [START_REF] Giffin | The Migraine Postdrome: An Electronic Diary Study[END_REF]. It is still unclear however whether these symptoms were initiated during the headache phase or appeared after due to the effect of the abortive medication as reported by some patients.

Episodic Migraine vs. Chronic Migraine

Approximately 3% of people with episodic migraine progress gradually to chronic migraine each year (A. I. Scher et al. 2003). Chronic migraine is prevalent in 1.4-2.2% of the worldwide population (1.7-4% for females and 0.6-0.7% for males) (Natoli et al. 2010). According to the ICHD-3, chronic migraine is characterized by 15 or more headache days per month, for a duration exceeding three months, which on at least eight days per month meets the criteria for migraine with or without aura (IHS 2018). Chronic patients report an increase in headache frequency, more frequent cutaneous hypersensitivities, and more robust nausea and photophobia (Figure 2) [START_REF] Rattanawong | Neurobiology of Migraine Progression[END_REF]. In Europe, the average medical costs of patients with chronic migraine are three times higher than those of episodic migraine as well as a twice-fold higher disability score [START_REF] Bigal | Assessment of Migraine Disability Using the Migraine Disability Assessment (MIDAS) Questionnaire: A Comparison of Chronic Migraine With Episodic Migraine[END_REF][START_REF] Bloudek | Cost of Healthcare for Patients with Migraine in Five European Countries: Results from the International Burden of Migraine Study (IBMS)[END_REF]). The pathophysiological mechanisms by which migraine transforms from an episodic to a chronic state are complex and poorly understood (a special chapter within this thesis is focused on these mechanisms).

Several factors have been associated with the risk of migraine progression, including non-modifiable (sex, hormones, and genetic background) to modifiable (acute headache medication use, obesity, other pain syndromes, and stress) factors (Ann I. [START_REF] Scher | Risk Factors for Headache Chronification[END_REF]. Headache frequency is also an important risk factor for progression. Individuals with ≥4 headache days per month have an exponential risk of migraine transformation. Moreover, a negative impact comes from migraine on many aspects of one's life with a higher burden among chronic migraineurs compared to those with episodic migraine [START_REF] Buse | Life With Migraine: Effects on Relationships, Career, and Finances From the Chronic Migraine Epidemiology and Outcomes (CaMEO) Study[END_REF]).

Risk Factors, Comorbidities, and Triggers

More than 70% of migraineurs associate their attacks with certain triggers, and this figure exceeds 95% when subjects are responding to a specific list of triggers [START_REF] Baldacci | Photophobia Is Associated with Allodynia during Migraine Attack[END_REF][START_REF] Kelman | The Triggers or Precipitants of the Acute Migraine Attack[END_REF]) (Figure 3). The most common triggers are stress (79.7%), hormones in women (65.1%), diet (57.3%), weather (53.2%), sleep disturbances (49.8%), and odors (43.7%) [START_REF] Kelman | The Triggers or Precipitants of the Acute Migraine Attack[END_REF]. Predisposing factors include also sex, stress susceptibility, and genetic background among others. In addition, the presence of concomitant chronic illnesses (e.g. mood, metabolic or inflammatory disorders) can play a bidirectional correlative link with migraine that brings additional elements to the table to further understand the pathophysiology of migraine.

Stress and Lifestyle

The stress response is generally associated with a subjective feeling of external or internal threat/demand and depends on the stressor quality, individual susceptibility, context, and previous experiences. It has been shown that psychological stress plays an important role not only before the onset of migraine but also in its maintenance and the frequency of attacks [START_REF] Seng | Lifestyle Factors and Migraine[END_REF]. Moreover, stressful life events may increase the risk of progression from episodic to chronic migraine (A. I. Scher et al. 2003). However, the distinction between stress as a trigger or as a migraineassociated symptom is not always clear for patients, and some longitudinal studies have revealed contradictory findings.

Lifestyle-related behavioral interventions, such as sleep, also impact migraine.

Indeed, poor quality sleep (tiredness, excessive sleep, and restless sleep) has commonly been associated with next-day headaches in people with migraine [START_REF] Peris | Towards Improved Migraine Management: Determining Potential Trigger Factors in Individual Patients[END_REF].

Like stress, the directionality of the sleep-migraine attack relationship is not clear, and poor sleep quality could be characterized as part of premonitory symptoms, such as fatigue and yawning. In addition, the surrounding environment (certain smells, strong lights, stroboscopic illuminations, blue LEDs, and alarming noises) can perturb lifestyle and sleep, and therefore, influence the frequency and severity of attacks [START_REF] Hougaard | Provocation of Migraine with Aura Using Natural Trigger Factors[END_REF]).

Genetic Background

Genetic background has been found to play a vital role in the development of migraine (H. [START_REF] Zhao | Gene-Based Pleiotropy across Migraine with Aura and Migraine without Aura Patient Groups[END_REF]. In monozygotic twins, the concordance rate is higher compared to dizygotic twins indicating that migraine is of a heritable nature with 42% of heritability [START_REF] Polderman | Meta-Analysis of the Heritability of Human Traits Based on Fifty Years of Twin Studies[END_REF]. Genome-wide association studies have been able

to identify 38 loci associated with a higher risk of migraine [START_REF] Gormley | Meta-Analysis of 375,000 Individuals Identifies 38 Susceptibility Loci for Migraine[END_REF], mainly localized in vascular tissue. Mutations have been also shown to affect the entire brain milieu (neurons, astrocytes, microglia, oligodendrocytes, and endothelial cells) indicating that all cell types play a role in migraine pathogenesis [START_REF] Renthal | Localization of Migraine Susceptibility Genes in Human Brain by Single-Cell RNA Sequencing[END_REF].

Sex Hormones

Ovarian hormones play a significant role in some adolescent girls and young adult women with migraine. The incidence of migraine is similar in both sexes until the age of 9 (2.5% of girls and 2.4% of boys) and then diverges to the disadvantage of girls [START_REF] Pavlovic | Sex-Related Influences in Migraine[END_REF]. During the female reproductive cycle, hormonal changes can influence or induce migraine. Onset of migraine frequently occurs around the time of menarche, as cyclic hormonal changes begin. The IHS defines "migraine attacks related to menstruation" as migraine without aura that occurs between days -2 and +3 of a menstrual cycle, with day 1 defined as the onset of menstrual flow [START_REF] Nierenburg | Systematic Review of Preventive and Acute Treatment of Menstrual Migraine[END_REF]. Up to 70% of female patients suffering menstrual-related migraine report more painful and longer headaches, and higher resistance to treatment than non-menstrualrelated migraines [START_REF] Bushman | Headaches Through a Woman's Life[END_REF][START_REF] Calhoun | Elimination of Menstrual-Related Migraine Beneficially Impacts Chronification and Medication Overuse[END_REF].

Menstrual-related migraine frequently coincides with a drop in plasma estrogen, which contributes to disease propagation [START_REF] Warnock | Hormone-Related Migraine Headaches and Mood Disorders: Treatment with Estrogen Stabilization[END_REF]. Adverse effects include decreased serotonergic and opioidergic tonus as well as increased excitability of trigeminal afferents, which, as we will see later, are involved in migraine initiation. (Aggarwal, Puri, and Puri 2012). Furthermore, estrogen decline is larger in migraine patients than in healthy controls [START_REF] Reddy | The Complex Relationship between Estrogen and Migraines: A Scoping Review[END_REF]). Hence, this evidence indicates that migraine is associated with changes in circulating estrogen. Nevertheless, how estrogen might be protective and why its decrease triggers attacks remain unanswered.

Mood Disorders and Cognition

Some epidemiological studies have suggested that migraine is bidirectionally associated with several other disorders, including anxiety and depression, in both men and women [START_REF] Breslau | Headache and Major Depression: Is the Association Specific to Migraine?[END_REF][START_REF] Zwart | Depression and Anxiety Disorders Associated with Headache Frequency. The Nord-Trøndelag Health Study[END_REF]. On one hand, the higher the headache frequency the more the levels of anxiety and depression [START_REF] Oh | Combination of Anxiety and Depression Is Associated with an Increased Headache Frequency in Migraineurs: A Population-Based Study[END_REF]). On the other hand, anxiety disorders are two to five times more frequent in patients with migraine [START_REF] Buse | Psychiatric Comorbidities of Episodic and Chronic Migraine[END_REF], and migraineurs are 2.5 times more likely to suffer from depression compared to non-migraineurs (R. B. Lipton et al. 2000;[START_REF] Minen | Migraine and Its Psychiatric Comorbidities[END_REF].

Migraine is also associated with poor cognitive performance [START_REF] Baena | Migraine and Cognitive Function: Baseline Findings from the Brazilian Longitudinal Study of Adult Health: ELSA-Brasil[END_REF]. Patients frequently attend consultations for subjective cognitive complaints that are mainly attention-and memory-related and appear during or after migraine attacks, and contribute to the disability of patients experiencing them. Cognitive symptoms came in second place to pain in terms of severity and attack-related impairment [START_REF] Gil-Gouveia | The Impact of Cognitive Symptoms on Migraine Attack-Related Disability[END_REF]. Severe subjective cognitive decline is associated with worse pain severity, enhanced levels of anxiety and depression, poor sleep quality, and decreased sleep duration [START_REF] Lee | Subjective Cognitive Decline in Patients with Migraine and Its Relationship with Depression, Anxiety, and Sleep Quality[END_REF].

Gut-Brain Axis

The terminology "gut-brain axis" points out a bidirectional network between the gastrointestinal system and the CNS (central nervous system), which consists of multiple connections, including the immune system and bacterial metabolism. The dysregulation of the gut-brain axis is associated with altered permeability of the bloodbrain barrier (BBB) and neuroinflammation and has been implicated in several neurological disorders such as multiple sclerosis, anxiety, depression, Alzheimer's, and

Parkinson's diseases [START_REF] Socała | The Role of Microbiota-Gut-Brain Axis in Neuropsychiatric and Neurological Disorders[END_REF].

Evidence also supports the involvement of the gut-brain axis in migraine. During the attack, patients suffer gastrointestinal symptoms such as nausea and emesis, constipation, or diarrhea. Migraine is frequently comorbid with metabolic disorders [e.g. celiac disease [START_REF] Zis | Headache Associated with Coeliac Disease: A Systematic Review and Meta-Analysis[END_REF]], gastrointestinal disorders [predominately inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS)], and chronic inflammatory diseases [rheumatoid arthritis and multiple sclerosis (X. [START_REF] Moisset | Migraine Prevalence in Inflammatory Bowel Disease Patients: A Tertiary-Care Centre Cross-Sectional Study[END_REF][START_REF] Moisset | Migraine Headaches and Pain with Neuropathic Characteristics: Comorbid Conditions in Patients with Multiple Sclerosis[END_REF]]. Moreover, patients with migraine have been described to present a microbiota dysbiosis, which was positively correlated with attack severity and duration [START_REF] Georgescu | Migraine without Aura and Subclinical Atherosclerosis in Young Females: Is Gut Microbiota to Blame?[END_REF]. Microbial alterations trigger gut barrier dysfunctions, that increase the permeability of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, and interleukins (IL) 1β and 6, affecting nociceptive responses in the trigeminal pathway during migraine initiation (as we will see later) [START_REF] Arzani | Gut-Brain Axis and Migraine Headache: A Comprehensive Review[END_REF]). In addition, microbiota dysbiosis contributes to chronicity in mice, since antibiotic treatment was shown to prolong the nitroglycerine (NTG)-induced acute migraine-like pain in wild-type mice, while probiotic administration significantly inhibited the pain prolongation (Yuanyuan [START_REF] Tang | Gut Microbiota Dysbiosis Enhances Migraine-Like Pain Via TNFα Upregulation[END_REF]. Taken together, evidence supports the pivotal action of the microbiome and gut-brain axis in migraine, although more research is needed. The modulatory role of inflammation in migraine pathogenesis will be explained more in-depth in another chapter.

Diet

The impact of diet and nutrition on headaches has been extensively debated.

Diet is believed to affect the modulation of neuroreceptors, neuropeptides, and CNS responses, and to play a role in causing and/or relieving inflammation (V. T. [START_REF] Martin | Diet and Headache: Part 2[END_REF]. Although long lists of potentially migraine-associated foods have been established (chocolate, citrus fruits, nuts, tomatoes, onions, caffeine, nitrites, gluten, red meat…) [START_REF] Nazari | Migraine and Its Relationship with Dietary Habits in Women[END_REF] controversy remains in the field: some dietary nutrients are considered as migraine triggers, while others may play a role in its prevention and prophylaxis. Moreover, exposure to a given food may not always trigger a headache, and the amount of food or the time of exposure might largely influence the outcome. Therefore, the identification of diet-associated modulators is challenging.

Food diaries and specific serological testing have been used to identify triggers in individual patients, with the aim of avoiding them and preventing migraine. This is the basis of elimination diet strategies. However, not only the diet content but also the meal pattern has been associated with migraine [START_REF] Bakırhan | Associations between Diet Quality, DASH and Mediterranean Dietary Patterns and Migraine Characteristics[END_REF]. Several types of diets, including ketogenic, high-folate, low-fat, modified Atkins, and high omega-3/low omega-6 diets, have been proposed to be beneficial (Razeghi [START_REF] Jahromi | Association of Diet and Headache[END_REF]). Many of these diets are not mechanism or evidencedriven, but have been proposed to act through a variety of mechanisms: on serotoninergic dysfunction, neuronal excitability, and/or concentration of migrainerelated substances [such as calcitonin gene-related peptide (CGRP) [START_REF] Fila | Nutrition and Calcitonin Gene Related Peptide (CGRP) in Migraine[END_REF] and nitric oxide (NO)], neuroinflammation, or hypothalamic function. Propositions have been done that dietary approaches with beneficial effects on the gut microbiota and gutbrain axis may improve migraine. For example, supplementation with vitamin D and omega-3 fatty acids, consumption of a low glycemic-index diet, probiotics, low-fat vegan diet, gluten-free diet, and weight-loss diets [START_REF] Arzani | Gut-Brain Axis and Migraine Headache: A Comprehensive Review[END_REF]. However, research on the microbiome and migraine is still novel, and future investigations are required before recommending a dietary intervention aiming to change the microbiome for migraine prevention.

II-Generalities of Pain Processing

According to the International Association for the Study of Pain (IASP), pain is always a personal experience that is influenced to varying degrees by biological, psychological, and social factors. Pain and nociception are different phenomena. While pain is defined as "an unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage" by the IASP, nociception is "the neural process of encoding noxious stimuli" [START_REF] Raja | The Revised International Association for the Study of Pain Definition of Pain: Concepts, Challenges, and Compromises[END_REF].

Noxious stimuli are detected by nociceptors, which are subpopulations of peripheral nerve fibers [START_REF] Basbaum | Cellular and Molecular Mechanisms of Pain[END_REF] 4). C-fibers are also divided into two contingents according to their neurochemical nature: some contain peptides, such as substance P or CGRP, and are referred to as peptidergic fibers; while others are devoid of them, but are capable of binding to a specific lectin, isolectin B4 (IB4), and are referred to as non-peptidergic fibers [START_REF] Dallel | Neurobiology of trigeminal pain][END_REF].

Nociceptors send noxious information to the dorsal horn of the spinal cord through the dorsal root ganglia and synapse with second-order neurons. The discrimination between noxious and non-noxious is done according to the gate control theory [START_REF] Melzack | Pain Mechanisms: A New Theory[END_REF][START_REF] Mendell | Constructing and Deconstructing the Gate Theory of Pain[END_REF], which is based on the balance of three simultaneous inputs into the convergence neurons located in the posterior dorsal horn:

nociceptive inputs from Aδand C-fibers; non-nociceptive inputs from Aα and Aβfibers; and inhibitory inputs from the descending inhibitory pathway. The selective stimulation of Aα/Aβ-fibers blocks Aδ/C fibers at the level of the substantia gelatinosa located in layer II. Like a gate, the cells of the substantia gelatinosa regulate the entry from the periphery to the convergence neurons and, therefore, control the access to the CNS. When the activity of the non-noxious fibers predominates, the activity of the noxious fibers is inhibited. However, when the activity of Aδ/C-fibers predominates, the nociceptive message is sent by the second-order neurons.

From there, the message is transmitted to supraspinal areas by projection neurons located in layers I and V, by several ascending pathways, which connect the spinal cord to the thalamus direct or indirectly [START_REF] Willis | Neuroanatomy of the Pain System and of the Pathways That Modulate Pain[END_REF] (Figure 4).

The message then reaches the cerebral cortex for integration purposes and emotional interpretation [START_REF] Julius | Molecular Mechanisms of Nociception[END_REF].

Descending inhibitory supraspinal modulation occurs through the brainstem, and more specifically through three major structures: the monoaminergic periaqueductal gray (PAG), the serotoninergic nucleus raphe magnus (NRM), and the noradrenergic locus coeruleus (LC). The stimulation of these structures exerts an inhibitory control on convergence neurons in the dorsal horn by blocking the transmission of nociceptive messages [START_REF] Ossipov | Central Modulation of Pain[END_REF].

III. Anatomical Substrates of Trigeminal Pain Pathways

The Trigeminovascular System

The trigeminovascular system integrates, in the brainstem, somatosensory information (mechanical, thermal, and proprioceptive) from the orofacial sphere and the meninges, and plays a pivotal role in the etiology of migraine and its associated symptoms. This system encompasses a rich plexus of small pseudo-unipolar sensory neurons that originate from the trigeminal ganglion and upper cervical dorsal nerve roots with axonal projections to meningeal blood vessels, including the superior sagittal sinus, middle meningeal artery, and large cerebral arteries [START_REF] Ray | Experimental Studies on Headache: Pain-Sensitive Structures of the Head and Their Significance in Headache[END_REF].

Stimulation of the dura mater with various stimuli (mechanical, chemical, or electrical) consequently leads to headache pain during a migraine attack (including the frontal, temporal, parietal, occipital, and high cervical regions) as well as other migraine symptoms such as nausea and photophobia [START_REF] Mcnaughton | Innervation of Intracranial Structures: A Reappraisal[END_REF][START_REF] Penfield | Dural Headache and Innervation of the Dura Mater[END_REF].

The Meninges

Meninges envelop the CNS, the brain and the spinal cord, the intracranial spinal cord, the intracranial portion of cranial nerves, and the spinal nerve roots. Their primary function is to protect and anchor the brain and supply a support system for the blood vessels, lymphatics, nerves, and cerebrospinal fluid that surrounds the CNS. The meninges comprise two separate layers, the dura mater, and the leptomeninges. The dura mater or pachymeninx is a thick layer of connective tissue while the leptomeninges can be further separated into the arachnoid and pia mater [START_REF] Haines | On the Question of a Subdural Space[END_REF]. Recently a fourth layer, named a subarachnoid lymphatic-like membrane, that compartmentalizes the subarachnoid space has been detected [START_REF] Møllgård | A Mesothelium Divides the Subarachnoid Space into Functional Compartments[END_REF].

Pain molecules have shown a robust expression at the level of the cranial dura, with the sensory peptides, CGRP, and substance P, being expressed in axons innervating the cranial dura, cranial arteries, and venous sinuses (L. [START_REF] Csati | Distribution of Vasoactive Intestinal Peptide, Pituitary Adenylate Cyclase-Activating Peptide, Nitric Oxide Synthase, and Their Receptors in Human and Rat Sphenopalatine Ganglion[END_REF][START_REF] Meßlinger | Innervation of the Dura Mater Encephali of Cat and Rat: Ultrastructure and Calcitonin Gene-Related Peptide-like and Substance P-like Immunoreactivity[END_REF]. The other meningeal layers, primarily the pia matter, also play a role in sensory processing [START_REF] Dallel | Neurobiology of trigeminal pain][END_REF][START_REF] Fontaine | Dural and Pial Pain-Sensitive Structures in Humans: New Inputs from Awake Craniotomies[END_REF]. Pial arteries are the main site of sensory (mediates conscious activities) and autonomic (mediates unconscious activities) fiber innervations originating from sympathetic, parasympathetic, and sensory trigeminal ganglia [START_REF] Levy | Current Understanding of Meningeal and Cerebral Vascular Function Underlying Migraine Headache[END_REF].

The Peripheral Trigeminal Afferents

Intracranial and meningeal vasculature inputs come from the trigeminal nerve's three extracranial divisions (V1: Ophthalmic, V2: Maxillary, and V3: Mandibular). The V1 branch is a relay of sensory information coming from facial regions located above the orbit of the eyes; the V2 branch is responsible for sensory information between the mouth and the orbit; and the V3 branch has both a motor and sensory component with only the latter projecting to the spinal trigeminal nucleus (Figure 4). These three divisions fuse at the trigeminal ganglion, and then extend as sensory and motor rootlets towards the different trigeminal nuclei, including the motor, principal sensory, spinal, and mesencephalic nuclei, afterward projecting towards the cortex [START_REF] Erzurumlu | Mapping the Face in the Somatosensory Brainstem[END_REF]. Adapted from [START_REF] Hu | Trigeminal Brainstem Nuclear Complex, Physiology[END_REF].

Trigeminal axons are of two natures, non-myelinated C-fibers, and thinly myelinated Aδ -fibers [START_REF] Pennisi | Histometric Study of Myelinated Fibers in the Human Trigeminal Nerve[END_REF]. Additionally, Aβ-fibers have been found.

Since no other sensation can be triggered at the meningeal level, they most likely also serve a nociceptive function [START_REF] Schueler | Innervation of Rat and Human Dura Mater and Pericranial Tissues in the Parieto-Temporal Region by Meningeal Afferents[END_REF]. Both peptidergic and nonpeptidergic C-fibers are expressed, just like at the level of the skin. As they are suspected to be implicated in the neurogenic inflammation thought to occur during migraine attacks, the former has received considerable attention.

The Spinal Trigeminal Nucleus

Located in the lateral medulla of the brainstem, the spinal trigeminal nucleus is a relay of multiple sensory modalities including pain, touch, and temperature from the ipsilateral facial side in addition to relaying nociceptive information from the supratentorial dura mater (T. [START_REF] Bartsch | Increased Responses in Trigeminocervical Nociceptive Neurons to Cervical Input after Stimulation of the Dura Mater[END_REF]. The spinal trigeminal nucleus is divided into three nuclei, the pars oralis, pars interporalis, and pars caudalis. The pars oralis is involved in stem reflexes, while the pars interpolaris receives input from the ipsilateral face, and the pars caudalis constitutes the ascending anterior trigeminothalamic tract [START_REF] Bradnam | The Role of the Trigeminal Sensory Nuclear Complex in the Pathophysiology of Craniocervical Dystonia[END_REF]. We primarily focus on the pars caudalis or the spinal trigeminal nucleus caudalis (Sp5c) that receives input from the face, cheek, forehead, and jaw and is a primary relay of nociceptive and thermal information (Barry J. [START_REF] Sessle | Acute and Chronic Craniofacial Pain: Brainstem Mechanisms of Nociceptive Transmission and Neuroplasticity, and Their Clinical Correlates[END_REF].

Organization of the Sp5c

The Sp5c is a portion of the spinal trigeminal nucleus that extends from the obex, the termination of the ependymal duct in the fourth ventricle, to the first cervical segments where it enters in continuity with the dorsal horn of the spinal cord. This structure shares various cytoarchitectural similarities with the posterior horn, hence named the medullary posterior horn. It is divided into five distinct laminae according to the size and morphology of neurons [START_REF] Rexed | The Cytoarchitectonic Organization of the Spinal Cord in the Cat[END_REF]) (Figure 5). Layer I termed the marginal layer, is the most superficial layer of the Sp5c [START_REF] Olszewski | On the Anatomical and Functional Organization of the Spinal Trigeminal Nucleus[END_REF]. It includes both small glutamatergic/glycinergic interneurons and larger projection neurons.

Neurons of lamina I are crucial in relaying nociceptive information from the peripheral nervous system toward higher-order regions of the brain. The latter being primarily the thalamus, hypothalamus, and parabrachial nucleus [START_REF] Saito | Ascending Projections of Nociceptive Neurons from Trigeminal Subnucleus Caudalis: A Population Approach[END_REF]. Layer II is also referred to as the substantia gelatinosa due to its translucent appearance courtesy of the low amount of myelinated fibers [START_REF] Olszewski | On the Anatomical and Functional Organization of the Spinal Trigeminal Nucleus[END_REF]. It is further divided into lamina II outer (IIo) and lamina II inner (IIi) with different roles in somatosensory processing.

This has been shown as the Aδ-fibers terminate in IIo only while the C-fibers terminate in both laminae IIo and IIi (L. [START_REF] Li | The Functional Organization of Cutaneous Low-Threshold Mechanosensory Neurons[END_REF]) (Figure 5). It also demonstrated the existence of substance P receptors in the postsynaptic neurons of both laminae I and II (D. [START_REF] Wang | γ-Aminobutyric Acid-and Glycine-Immunoreactive Neurons Postsynaptic to Substance P-Immunoreactive Axon Terminals in the Superficial Layers of the Rat Medullary Dorsal Horn[END_REF]. In addition, the marker protein kinase C (PKC)γ, is primarily Layer III encompasses a high density of both interneurons and projection neurons larger in size compared to lamina II. Neurons of lamina III are activated by vibrissae and/or guard hair deflection [START_REF] Renehan | Structure-Function Relationships in Rat Medullary and Cervical Dorsal Horns. II. Medullary Dorsal Horn Cells[END_REF]. Layers IV-V are separated from lamina II by the heterogeneity in the neuronal size of lamina IV neurons compared to the smaller cells of lamina III. In addition, there is extensive evidence that identified lamina V neurons as important in pain pathways [START_REF] Amano | Responses of Neurons in Feline Trigeminal Subnucleus Caudalis (Medullary Dorsal Horn) to Cutaneous, Intraoral, and Muscle Afferent Stimuli[END_REF].

Central Afferent Projections

The trigeminocervical complex (TCC) encompasses the Sp5C and the C1 and C2 regions of the cervical spinal cord. The stimulation of the meningeal vasculature has been shown to result in the activation of the TCC [START_REF] Goadsby | Differential Effects of Low Dose CP122,288 and Eletriptan on Fos Expression Due to Stimulation of the Superior Sagittal Sinus in Cat☆[END_REF]. Such activated neural pathways have been pivotal to further deciphering the pathophysiology of migraine and finding potential therapeutic targets. From the TCC, nociceptive messages are transmitted to the cerebral cortex, the amygdala, or the hypothalamus via relays located in the thalamus, the bulbar reticular formation, the parabrachial nucleus, or the nucleus of the solitary tract (Figure 6). These structures participate in the integration of the different aspects of pain.

In the thalamus, the nature of the message is defined by the thalamic nuclei of origin. The ventral posteromedial (VPM) thalamic nucleus for instance contains durasensitive neurons having a role in the sensory-discriminative components of migraine (location, intensity, and quality of pain), which project towards the insula as well as primary and secondary somatosensory cortices (S1/S2) [START_REF] Noseda | Cortical Projections of Functionally Identified Thalamic Trigeminovascular Neurons: Implications for Migraine Headache and Its Associated Symptoms[END_REF]. The durasensitive neurons of the posterior (Po), lateral posterior/dorsal (LP/LD) thalamic nuclei have a role in motor, focus, cutaneous hypersensitivity (allodynia), phonophobia, and photophobia and hence project to motor, somatosensory, auditory, visual, olfactory, and retrosplenial cortices.

IV. Modulation of the Trigeminal Pain Pathways

Several descending pathways modulate trigeminovascular nociception processing (Figure 6). Starting with the brainstem modulation, the PAG and the LC, located in the midbrain, makes direct projections with the trigeminovascular system. For the LC modulation, a separate section will be addressed. The PAG, could either augment the neuronal activity facilitating trigeminovascular pain transmission or suppress the activity of neurons in the spinal and medullary dorsal horn that inhibit trigeminovascular pain transmission [START_REF] Porreca | Chronic Pain and Medullary Descending Facilitation[END_REF]. The RVM (rostral ventromedial medulla) is a cluster of neurons in the medulla that receives bidirectional projections from the PAG and the spinal dorsal horn. Descending modulatory cells of the RVM either augments or blocks the responses of ascending nociceptive neurons to noxious stimulation of their respective receptive fields [START_REF] Edelmayer | Medullary Pain Facilitating Neurons Mediate Allodynia in Headache-Related Pain[END_REF].

The hypothalamus, a key player in autonomic and endocrine control, has been implicated in migraine, precisely in the premonitory symptoms experienced before an attack (sleep-wake cycle disturbances and mood changes) (Marie [START_REF] Denuelle | Hypothalamic Activation in Spontaneous Migraine Attacks[END_REF]; N. J. [START_REF] Giffin | Premonitory Symptoms in Migraine: An Electronic Diary Study[END_REF]. There is a reciprocal anatomical connection between the hypothalamus and Sp5c [START_REF] Malick | Trigeminohypothalamic and Reticulohypothalamic Tract Neurons in the Upper Cervical Spinal Cord and Caudal Medulla of the Rat[END_REF]. Direct projections toward the spinal and trigeminal dorsal horns and indirect projections are received by nociceptive brainstem structures (PAG, LC, and RVM) from various hypothalamic areas [START_REF] Holstege | Some Anatomical Observations on the Projections from the Hypothalamus to Brainstem and Spinal Cord: An HRP and Autoradiographic Tracing Study in the Cat[END_REF][START_REF] Robert | Paraventricular Hypothalamic Regulation of Trigeminovascular Mechanisms Involved in Headaches[END_REF]. The hypothalamus has a role in several aspects of migraine, according to structural connections between it and Sp5c and the existence of neurons that produce c-Fos in numerous hypothalamic nuclei following dural stimulation (Rami [START_REF] Burstein | Unitary Hypothesis for Multiple Triggers of the Pain and Strain of Migraine[END_REF]. Hypothalamic activation during migraine is also consistent with a role in pain modulation and hence may contribute to the development of central sensitization of trigeminovascular neurons. A recent study in rodents has shown experimental evidence for this scenario whereby the paraventricular hypothalamic nucleus (PVN) directly controls both spontaneous and evoked activities of the Sp5c [START_REF] Robert | Paraventricular Hypothalamic Regulation of Trigeminovascular Mechanisms Involved in Headaches[END_REF]. Therefore, PVN neurons play a role as either modulators or triggers of migraine through the integration of nociceptive, autonomic, and stress responses. Moreover, the contribution to the parasympathetic autonomic symptoms observed in migraine is done by the circuit projecting from the hypothalamus to the superior salivatory nuclei in the brainstem [START_REF] Lai | Cranial Autonomic Symptoms in Migraine: Characteristics and Comparison with Cluster Headache[END_REF].

The thalamus is a major relay of sensory nociceptive information to be processed in respective cortical structures. Its role in migraine goes beyond the relay part and extends toward the modulation of trigeminovascular and other spinal nociceptive inputs. The thalamic nuclei VPM, Po, and LP/LD receive nociceptive inputs from the dura mater and hence can modulate the perception of pain (Rami [START_REF] Burstein | Thalamic Sensitization Transforms Localized Pain into Widespread Allodynia[END_REF]Younis et al. 2019). Sensitization, which is an increase in the responsiveness of nociceptive neurons to their normal afferent input, of the thalamic neurons (precisely the VPM and lateral nuclei) leads to the spreading of cutaneous hypersensitivity (or cutaneous allodynia) (Rami [START_REF] Burstein | Thalamic Sensitization Transforms Localized Pain into Widespread Allodynia[END_REF]. It affects 60-80% of migraineurs (Rami Burstein, Collins, and Jakubowski 2004), in both cephalic and extracephalic (hands, feet) areas. The successional sensitization of first, second, and third, trigeminovascular neurons through the signals coming from the peripheral dural trigeminal innervations is thought to be the reason for the sensitization of the thalamic nuclei (Rami [START_REF] Burstein | Chemical Stimulation of the Intracranial Dura Induces Enhanced Responses to Facial Stimulation in Brain Stem Trigeminal Neurons[END_REF][START_REF] Burstein | Thalamic Sensitization Transforms Localized Pain into Widespread Allodynia[END_REF][START_REF] Strassman | Sensitization of Meningeal Sensory Neurons and the Origin of Headaches[END_REF]. The thalamus's involvement in migraine, precisely photophobia is clear.

Photophobia is one of the principal non-head pain symptoms of migraine. Primarily defined as an abnormal sensitivity to light, other definitions arose as well, such as ocular discomfort (oculodynia), exacerbation of headache by light, and a general aversion to light (Fine and Digre 1995;Lebensohn 1951;[START_REF] Noseda | A Neural Mechanism for Exacerbation of Headache by Light[END_REF]Noseda, Borsook, and Burstein 2017). In a recent mouse model, the stimulation of the Po nucleus was sufficient to induce light-aversive behavior (Sowers et al. 2020). It is important to note that the neural pathways that mediate the aversion to light are different than the ones mediating the exacerbation of headache by light or the ones mediating ocular discomfort [START_REF] Noseda | Current Understanding of Photophobia, Visual Networks and Headaches[END_REF].

Trigeminal nociceptive modulation originates from the cortex. The mechanisms by which alterations in cortical excitability contribute to migraine pathophysiology are largely unknown; however, through cortico-trigeminal connections, distinct cortical regions and their level of excitability may be implicated in the regulation of migraine pain. Direct descending projections have been described from the cerebral cortex to the Sp5c in both rodents and humans [START_REF] Kuypers | Corticobular Connexions to the Pons and Lower Brain-Stem in Man: An Anatomical Study[END_REF][START_REF] Noseda | Changes of Meningeal Excitability Mediated by Corticotrigeminal Networks: A Link for the Endogenous Modulation of Migraine Pain[END_REF] (Figure 7). The cortico-trigeminal projections originate mainly from the contralateral primary somatosensory and insular cortices and innervate both deep and superficial layers of the Sp5c. Anatomical positioning of these organized cortico-trigeminal networks aims at influencing meningeal nociception as what was demonstrated by S1mediated inhibition and insula-mediated facilitation of the excitability of Sp5c durasensitive neurons [START_REF] Noseda | Changes of Meningeal Excitability Mediated by Corticotrigeminal Networks: A Link for the Endogenous Modulation of Migraine Pain[END_REF][START_REF] Sessle | Functional Properties of Neurons in Cat Trigeminal Subnucleus Caudalis (Medullary Dorsal Horn). II. Modulation of Responses to Noxious and Nonnoxious Stimuli by Periaqueductal Gray, Nucleus Raphe Magnus, Cerebral Cortex, and Afferent Influences, and Effect of Naloxone[END_REF].

V. Migraine Mechanisms and Pathophysiology

Although understanding migraine attack pathophysiology has substantially improved, the pathogenesis of this disease (that is, why and how attacks are repeatedly triggered or occur spontaneously) remains poorly understood. Clinical and preclinical models (which are listed in a separate section below) have provided critically important insights into migraine mechanisms, but results from these studies must always be validated against those in spontaneous attacks.

The Premonitory Phase and Triggering Mechanisms

The Vascular Theory

Suggestions that migraine may have a vascular origin came from its pulsatile properties. Galen of Pergamon was the first to postulate the vascular theory in the second century [START_REF] Isler | The Galenic Tradition and Migraine[END_REF], which took hold when Ray and Wolf noted during the 1940s that the origin of migraine attacks was located in cerebral blood vessels [START_REF] Ray | Experimental Studies on Headache: Pain-Sensitive Structures of the Head and Their Significance in Headache[END_REF]. More studies showed that vasodilatory peptides, such as CGRP, NO, and PACAP, are all potent contributors to migraine pathophysiology [START_REF] Kuburas | Shared and Independent Roles of CGRP and PACAP in Migraine Pathophysiology[END_REF][START_REF] Pradhan | Targeted Nitric Oxide Synthase Inhibitors for Migraine[END_REF]. However, not all data fit this hypothesis.

In a cohort of 20 migraine patients, Ahn did not find a temporal relationship between arterial pulsation and throbbing migraine pain [START_REF] Ahn | On the Temporal Relationship Between Throbbing Migraine Pain and Arterial Pulse[END_REF]. Moreover, studies using NTG, a potent NO donor, have reported a lack of vasodilation during the headache phase [START_REF] Thomsen | A Nitric Oxide Donor (Nitroglycerin) Triggers Genuine Migraine Attacks[END_REF]. CGRP-evoked vasodilation couldn't induce a nociceptive effect on meningeal nociceptors [START_REF] Levy | Calcitonin Gene-Related Peptide Does Not Excite or Sensitize Meningeal Nociceptors: Implications for the Pathophysiology of Migraine[END_REF]. In addition, although Olesen mentioned in 1981 that the initial vasodilatory phase could be important in the pain process, pain ensued only after the subsidence of vasodilation [START_REF] Olesen | Focal Hyperemia Followed by Spreading Oligemia and Impaired Activation of Rcbf in Classic Migraine[END_REF]. The inducer of arterial vasodilation, Vasoactive intestinal peptide (VIP) also did not trigger a migraine attack in patients [START_REF] Rahmann | Vasoactive Intestinal Peptide Causes Marked Cephalic Vasodilation, but Does Not Induce Migraine[END_REF]). Amin et al. showed using MR-angiography that middle cerebral arterial dilation during spontaneous attacks persisted after relief with sumatriptan (an abortive drug; further discussed in another section), whereas extracranial arteries constricted [START_REF] Amin | Magnetic Resonance Angiography of Intracranial and Extracranial Arteries in Patients with Spontaneous Migraine without Aura: A Cross-Sectional Study[END_REF]. These data suggest that cerebral arterial dilation is an epiphenomenon reflecting activation of perivascular afferents in response to vasodilator neurotransmitters and support that vasculature is important in migraine but not the only player.

The Neuronal Theory

Dysfunction of the Brainstem

Studies conducted over the past decade suggest that the triggering of migraine attacks may be based on dysfunction of brainstem nuclei involved in modulatory control [START_REF] Afridi | A PET Study Exploring the Laterality of Brainstem Activation in Migraine Using Glyceryl Trinitrate[END_REF][START_REF] Moulton | Interictal Dysfunction of a Brainstem Descending Modulatory Center in Migraine Patients[END_REF]. Indeed, migraine attacks are accompanied by increased blood flow in various regions of the brainstem, including the PAG, LC, and NRM [START_REF] Afridi | A PET Study Exploring the Laterality of Brainstem Activation in Migraine Using Glyceryl Trinitrate[END_REF][START_REF] Bahra | Brainstem Activation Specific to Migraine Headache[END_REF]. Interestingly, a persistence of brain stem activity was documented in humans even after cessation of the headache and its confounding symptoms post-sumatriptan administration (Weiller et al. 1995). Hence, suggestions came about that there are headache-generating centers located in the brainstem.

Involvement of the PAG

The involvement of the PAG in the pathophysiology of migraine stems from clinical observations showing that in humans, the stimulation of the ventrolateral PAG in the treatment of chronic pain triggers migraine-like headaches [START_REF] Veloso | Headache Secondary to Deep Brain Implantation[END_REF]. Furthermore, the connections shared between the PAG and several brain areas involved in pain modulation, such as the prefrontal and anterior cingulate cortex, as well as the amygdala, are diminished, even between attacks, in migraine patients (Z. Chen et al. 2017;[START_REF] Mainero | Altered Functional Magnetic Resonance Imaging Resting-State Connectivity in Periaqueductal Gray Networks in Migraine[END_REF]. At the same time, stronger increased connections are formed with other areas such as the insula and other frontal and temporal regions (Schwedt et al. 2014). The involvement of PAG in migraine is also demonstrated through animal studies. Indeed, in various animal models, electrical stimulation [START_REF] Knight | The Periaqueductal Grey Matter Modulates Trigeminovascular Input: A Role in Migraine?11Presented in Part at the Xth International Headache Congress[END_REF] or microinjection of a triptan (anti-migraine medication) (T. [START_REF] Bartsch | Activation of 5-HT1B/1D Receptor in the Periaqueductal Gray Inhibits Nociception[END_REF]) into the PAG is sufficient to inhibit meningeal nociception.

Involvement of the Hypothalamus

Migraine's premonitory symptoms are highly associated with homeostatic functions that are regulated by the hypothalamus (feeding, sleep, and arousal). Through brain imaging studies, using positron emission tomography, the hypothalamus shows increased blood flow during the early stages of sudden migraine attacks (M. [START_REF] Denuelle | A PET Study of Photophobia during Spontaneous Migraine Attacks[END_REF], and in NTG-induced migraine attacks, changes are seen during the premonitory phase (Maniyar et al. 2014). In animals, stimulation of the rat dura mater has been shown to induce c-Fos protein expression in the ventromedial, dorsomedial, and paraventricular nuclei of the hypothalamus [START_REF] Malick | A Neurohistochemical Blueprint for Pain-Induced Loss of Appetite[END_REF], while stimulation of the cat's superior longitudinal sinus induces expression of this marker in the supraoptic and posterior nuclei of the hypothalamus [START_REF] Benjamin | Hypothalamic Activation after Stimulation of the Superior Sagittal Sinus in the Cat: A Fos Study[END_REF].

Dopaminergic areas in the hypothalamus precisely the A11 nucleus has shown to project toward the TCC and hence modulate the ascending trigeminovascular pathway [START_REF] Charbit | Trigeminocervical Complex Responses after Lesioning Dopaminergic A11 Nucleus Are Modified by Dopamine and Serotonin Mechanisms[END_REF]. In addition to dopamine, the hypothalamus synthesizes orexins, neuropeptides involved in arousal, appetite, and wakefulness, and controls the release of orexins from the pituitary gland. In a rat model of migraine, intravenous injections of an orexinergic receptor antagonist decrease meningeal vasodilation and TCC activation [START_REF] Hoffmann | Evidence for Orexinergic Mechanisms in Migraine[END_REF], indicating a pronociceptive role for orexins in this pathology. Thus, dysfunction of this inhibitory control could have a pro-nociceptive effect on the TCC, leading to the nociceptive phase of migraine [START_REF] Charbit | Trigeminocervical Complex Responses after Lesioning Dopaminergic A11 Nucleus Are Modified by Dopamine and Serotonin Mechanisms[END_REF].

Involvement of the LC

Several recent studies have confirmed the involvement of LC in the pathophysiology of migraine. This will be further explained in a separate chapter specified for the LC.

Cortical Hyperexcitability

The hypothesis of hyperexcitability of the cerebral cortex arose from the vulnerability of migraine sufferers to certain factors, such as stress or intense sensory stimuli, particularly visual, which trigger attacks. Generally speaking, the sensory cortex of migraine sufferers responds excessively to repeated stimuli, and this sensitivity sometimes persists permanently [START_REF] Coppola | Is the Cerebral Cortex Hyperexcitable or Hyperresponsive in Migraine?[END_REF].

Transcranial magnetic stimulation, suggests that there are alterations in the cortical activity precisely at the level of the occipital lobe which appears hyperactive in migraineurs (S. K. [START_REF] Aurora | The Occipital Cortex Is Hyperexcitable in Migraine; Evidence from TMS, FMRI and MEG Studies (Wolff Award[END_REF]. Moreover, visual cortex hyperexcitability was linked to photophobia during spontaneous migraine attacks (M. [START_REF] Denuelle | A PET Study of Photophobia during Spontaneous Migraine Attacks[END_REF]).

These observations suggest the existence of periodic changes in cortical excitability, which can lead to a migraine attack when the change in the cortical excitability threshold coincides with a triggering stimulus. These changes also touch the motor cortex [START_REF] Brighina | Abnormal Facilitatory Mechanisms in Motor Cortex of Migraine with Aura[END_REF]) and the parietal cortex [START_REF] Andreatta | Altered Processing of Emotional Stimuli in Migraine: An Event-Related Potential Study[END_REF]). The mechanisms underlying altered cortical excitability remain largely unknown and are probably multifactorial. The possibility of excessive excitation caused by an abnormal release of excitatory neurotransmitters is suggested by the finding of higher cerebrospinal fluid glutamate concentrations in chronic migraine sufferers (M. [START_REF] Peres | Cerebrospinal Fluid Glutamate Levels in Chronic Migraine[END_REF].

Cortical Spreading Depression and Migraine with Aura

Cortical Spreading Depression (CSD) or spreading depolarization is a transient wave of cortical neuronal depolarization. It is known to be the pathophysiological brain mechanism underlying the clinical phenomenon of migraine with aura (A. C. [START_REF] Charles | Cortical Spreading Depression and Migraine[END_REF]. The majority of cells in the brain undergo an almost full membrane depolarization during CSD, whereby a massive efflux of potassium and glutamate, and other neurotransmitters and neuromodulators in addition to an influx of H2O, sodium, and calcium coupled with neuronal swelling [START_REF] Somjen | Mechanisms of Spreading Depression and Hypoxic Spreading Depression-Like Depolarization[END_REF]. Metabolic and electrocortical changes during CSD are accompanied by changes in perfusion resulting in cortical blood flow changes (Kevin C. [START_REF] Brennan | Distinct Vascular Conduction With Cortical Spreading Depression[END_REF]). The actual mechanism by which CSD starts and ends are unknown. During a migraine aura in humans, there is no demonstration of an electrophysiological correlate of a CSD probably due to the difficulty in capturing a CSD in a non-invasive manner in such an unpredictable disorder. However, it has been profoundly shown in animal models and human brain direct recordings after trauma and ischemia [START_REF] Dreier | Cortical Spreading Ischaemia Is a Novel Process Involved in Ischaemic Damage in Patients with Aneurysmal Subarachnoid Haemorrhage[END_REF][START_REF] Lauritzen | Clinical Relevance of Cortical Spreading Depression in Neurological Disorders: Migraine, Malignant Stroke, Subarachnoid and Intracranial Hemorrhage, and Traumatic Brain Injury[END_REF].

The long debate about whether CSD is a migraine attack trigger is still ongoing without consensus. In terms of connection, it has been reported that CSD activates the trigeminal nociceptive pathways hence triggering a migraine attack [START_REF] Noseda | Changes of Meningeal Excitability Mediated by Corticotrigeminal Networks: A Link for the Endogenous Modulation of Migraine Pain[END_REF]X. Zhang et al. 2011). A CSD has been shown to activate both pial and dural macrophages and dendritic cells [START_REF] Schain | Activation of Pial and Dural Macrophages and Dendritic Cells by Cortical Spreading Depression[END_REF], and is associated with the release of nociceptive mediators [START_REF] Schain | Cortical Spreading Depression Closes Paravascular Space and Impairs Glymphatic Flow: Implications for Migraine Headache[END_REF]. Multiple exogenous and endogenous factors such as hormones (estrogen and testosterone), and medications (anti-migraine preventive drugs) also modulate brain tissue susceptibility in animal models of CSD [START_REF] Bogdanov | Migraine Preventive Drugs Differentially Affect Cortical Spreading Depression in Rat[END_REF][START_REF] Charles | Cortical Spreading Depression and Migraine[END_REF].

Mechanisms of the Headache Phase

The headache phase of migraine involves the ascending trigemino-thalamic pathway's activation. Based on early observations in humans who underwent awake brain surgery, the idea that pain during a migraine attack is perceived to be felt on intracranial structures (dura matter and intracranial vasculature) became well established [START_REF] Ray | Experimental Studies on Headache: Pain-Sensitive Structures of the Head and Their Significance in Headache[END_REF].

Neurogenic Inflammation

Data from Ray and Wolff added to consolidate the theory that the activation of nociceptive nerve fibers (C-and Aδ-fibers) innervating blood vessels could lead to a migraine attack [START_REF] Ray | Experimental Studies on Headache: Pain-Sensitive Structures of the Head and Their Significance in Headache[END_REF]. The activation of these fibers was an enigma until the theory of dural inflammation came into place [START_REF] Moskowitz | The Neurobiology of Vascular Head Pain[END_REF]. The latter is based on the local dural release of endogenous inflammatory mediators (CGRP, substance P, prostaglandins, and neurokinin A) hence inducing an increase in the blood flow (as described before), mast cell degranulation, leakage of plasma proteins, and aggregation of platelets. Mast cells, in turn, release inflammatory molecules, including histamine known to activate C-fibers and promote the release of CGRP [START_REF] Lassen | CGRP May Play a Causative Role in Migraine[END_REF].

Substance P, mainly from C-fibers, is a potent vasodilator and provokes the release of bradykinin, histamine, and serotonin (also called 5-hydroxytryptamine (5-HT)), contributing to inflammation. CGRP, also released by C-fibers, is a neuropeptide able to trigger headaches in migraineurs [START_REF] Lassen | CGRP May Play a Causative Role in Migraine[END_REF] It targets the CGRP receptor present in the blood vessel muscles, A-fibers, and glial cells, and induces brain vessel vasodilation. At the level of the trigeminal ganglion, 70% of CGRP-positive neurons co-localize with the TRPV1 receptors, more than in the dorsal horn, which could explain the pivotal role of CGRP in the trigeminovascular system signaling [START_REF] Price | Critical Evaluation of the Colocalization Between Calcitonin Gene-Related Peptide, Substance P, Transient Receptor Potential Vanilloid Subfamily Type 1 Immunoreactivities, and Isolectin B4 Binding in Primary Afferent Neurons of the Rat and Mouse[END_REF]. Regarding prostaglandins, they have a vasodilator activity and can evoke CGRP release. Their intravenous injection in controlled individuals triggers headaches. The sympathetic fibers will release 5-HT, which promotes the nociceptor excitation at the level of meninges and their respective sensitization (A. M. [START_REF] Strassman | Sensitization of Meningeal Sensory Neurons and the Origin of Headaches[END_REF], and NO, increases the synthesis and release of CGRP, in addition to its vasodilator activity. Thus, all of these pro-inflammatory molecules act synergistically to lower the response threshold of nociceptors to mechanical stimuli.

Peripheral and Central Sensitization

Following all that has been postulated about migraine, migraine is now considered a neurovascular disorder. This theory is based on the sensitization of peripheral and central trigeminovascular neurons. The throbbing perception of the headache is mediated by peripheral sensitization (A. M. [START_REF] Strassman | Sensitization of Meningeal Sensory Neurons and the Origin of Headaches[END_REF], while the sensitization of second-order neurons in the Sp5C mediates cephalic cutaneous hypersensitivity as well as muscle tenderness (R. [START_REF] Burstein | The Development of Cutaneous Allodynia during a Migraine Attack Clinical Evidence for the Sequential Recruitment of Spinal and Supraspinal Nociceptive Neurons in Migraine[END_REF][START_REF] Burstein | Thalamic Sensitization Transforms Localized Pain into Widespread Allodynia[END_REF]) (Figure 7). Indeed, systemic administration of NTG (S. Wu et al. 2022) or ISDN (Isosorbide dinitrate) (Flores Ramos et al. 2017) in rats, induced Sp5C sensitization associated with migraine-like pain. Moreover, in a mouse model of inflammatory soup, the astrocytic activation of the Sp5c contributed to the central sensitization in chronic migraine (L. [START_REF] Zhang | Temporal Characteristics of Astrocytic Activation in the TNC in a Mice Model of Pain Induced by Recurrent Dural Infusion of Inflammatory Soup[END_REF].

The expansion of the dura and cutaneous receptive fields, as well as enhanced responsiveness to mechanical and thermal stimulation of the skin and the dura, are all signs of this neuronal sensitization (Rami [START_REF] Burstein | Chemical Stimulation of the Intracranial Dura Induces Enhanced Responses to Facial Stimulation in Brain Stem Trigeminal Neurons[END_REF]. Trigemino-cervical neurons receiving inputs from both intracranial dura and extracranial periorbital skin are subjected to long-lasting sensitization following the topical application of inflammatory agents to the rat dura [START_REF] Ebersberger | Recordings From Brain Stem Neurons Responding to Chemical Stimulation of the Subarachnoid Space[END_REF][START_REF] Schepelmann | Activation of Trigeminal Brain Stem Neurons by Chemical Stimulation of the Dura Mater Encephali-Preparation for Studying Meningeal Nociception in the Rat: Eine Präparation Zum Studium Der Meningealen Nozizeption Bei Der Ratte[END_REF]. A malfunction in the pain-modulating systems may also be a factor in the development of central sensitization and the vulnerability to developing chronic migraine. The onset or maintenance of sensitization and the emergence of chronic migraine may both be influenced by an imbalance of pain facilitation and inhibition (Schwedt 2014;[START_REF] Welch | Periaqueductal Gray Matter Dysfunction in Migraine and Chronic Daily Headache May Be Due to Free Radical Damage[END_REF]). What's intriguing is that medications like the recently developed monoclonal antibodies against the CGRP system and botulinum toxin, which act peripherally at least on trigeminal fibers, can block chronic migraine in at least 60-70% of individuals. As peripheral sensory inputs are crucial for maintaining central drive in chronic migraine, this further supports the trigeminal system's crucial function. The proposition for a neural substrate for extracephalic cutaneous hypersensitivity during migraine wasn't done until recently. A recent study demonstrated that innocuous heat and brush stimuli induce a heightened activity in the pulvinar thalamic nucleus of patients showcasing signs of whole-body allodynia (cannot wear tight clothing, cannot use a heavy blanket, and cannot take a shower) during migraine, as compared to the pain-free state (Rami [START_REF] Burstein | Thalamic Sensitization Transforms Localized Pain into Widespread Allodynia[END_REF]). In addition, sensitization of thalamic trigeminovascular neurons located in VPM, Po, and LP nuclei occurred after the topical application of inflammatory molecules on the rat meninges (Rami [START_REF] Burstein | Thalamic Sensitization Transforms Localized Pain into Widespread Allodynia[END_REF]) (Figure 7). Altogether, it is suggested that the rostral subdivision of the pulvinar in the posterior thalamus of human beings and the most dorsal and posterior part of the thalamus (i.e., Po) in animals mediate to a certain extent whole-body cutaneous hypersensitivity.

The Interictal Migraine "Brain State"

Between migraine attacks, the brain of migraineurs exhibits structural and functional differences compared with the brain of healthy controls. Altered morphologies include changes in the grey and white matter volume and integrity, which are associated with functional interictal alterations including enhanced visual, auditory, somatosensory, and motor-evoked responses, enhanced nociceptive processing, and abnormal pain processing in the brainstem. Such alterations offer an alternative hypothesis that considers migraine as a "brain state" [START_REF] Goadsby | Pathophysiology of Migraine: A Disorder of Sensory Processing[END_REF]). The brainstem is in close relations with other regions and nuclei of the brain and hence any dysfunction in said regions alters the descending pain modulatory pathways. The latter leads to a false perception of pain under normal conditions and ongoing dysfunction further leads to the central sensitization of trigeminovascular neurons in addition to increasing both cutaneous extracephalic and cephalic allodynia [START_REF] Goadsby | Pathophysiology of Migraine: A Disorder of Sensory Processing[END_REF].

The same dysfunction could affect the hypothalamus and hence the sleep cycle, and the thalamus in terms of the hypersensitivity to smells, sounds, and light as it projects towards the cortex. Determining whether these structural and functional abnormalities are a result or a cause of the repetitive migraine attacks is critical to improving understanding of migraine pathophysiology.

Migraine as a Chronic Evolutive Condition

Genetic and Epigenetic Components

Although genome-wide association studies failed to shed light on the molecular alterations that are responsible for migraine's evolutive nature, one can postulate that knowledge from multiple variants will highlight which molecular pathways that could be involved in migraine pathophysiology (Van Den Maagdenberg, Nyholt, and Anttila 2019). 44 SNPs (single nucleotide polymorphism) were linked to migraine without aura in the most recent genome-wide association study, which involved samples from more than 300,000 controls and approximately 60,000 patients [START_REF] Gormley | Meta-Analysis of 375,000 Individuals Identifies 38 Susceptibility Loci for Migraine[END_REF] [START_REF] Fusco | Haploinsufficiency of ATP1A2 Encoding the Na+/K+ Pump Alpha2 Subunit Associated with Familial Hemiplegic Migraine Type 2[END_REF]. High-frequency discharges may be facilitated by SCN1A mutations, which may also raise glutamate levels in the synapses [START_REF] Dichgans | Mutation in the Neuronal Voltage-Gated Sodium Channel SCN1A in Familial Hemiplegic Migraine[END_REF]. In this way, glutamatergic synapses' neurons can fire more often than they would under normal circumstances, which could account for why migraine aura sufferers are more susceptible to CSD (Van Den Maagdenberg, Nyholt, and Anttila 2019). It's interesting to note that mice with the CACNA1A mutation have an altered CGRP expression and attenuated trigeminovascular nociceptive responses [START_REF] Mathew | Pathophysiology of Chronic Migraine and Mode of Action of Preventive Medications[END_REF][START_REF] Park | Differential Trigeminovascular Nociceptive Responses in the Thalamus in the Familial Hemiplegic Migraine 1 Knock-in Mouse: A Fos Protein Study[END_REF].

Only a few studies have been conducted on migraine patients when it comes to the emerging field of epigenetics. A small pilot study compared the genome-wide DNA methylation levels in episodic migraineurs and chronic migraineurs with medication overuse headache (MOH) before and after a detox program to identify changes in DNA methylation associated with headache chronification. Although there were no statistically significant differences between the groups at the various time points, specific CpG sites of interest were detected and are believed to be implicated in the comorbidity of neuropsychiatric disorder and drug addiction pathways [START_REF] Terlizzi | Epigenetic DNA Methylation Changes in Episodic and Chronic Migraine[END_REF]). These preliminary findings suggest that epigenetic processes play a role in migraine, and they may also play a role in the mechanisms underlying brain plasticity and other processes specific to migraine. All these preliminary results need to be replicated, though, because migraine, in its episodic and chronic forms, is a complex and multidimensional condition.

Brain Alterations

Beyond functional alterations, other investigations comparing migraine patients and controls have found variations in the structural integrity of the brain, affecting both the white and gray matter, that change over time. White-matter abnormalities were discovered in the brainstem and cerebellum of chronic migraine patients [START_REF] Bilgiç | Volumetric Differences Suggest Involvement of Cerebellum and Brainstem in Chronic Migraine[END_REF]. Various brain regions known to have a role in multisensory integration, nociception/anti-nociception, and analgesic reliance were shown to exhibit minor changes in gray matter volume in association with chronic migraine, according to other research (Gianluca [START_REF] Coppola | Cerebral Gray Matter Volume in Patients with Chronic Migraine: Correlations with Clinical Features[END_REF]. Gray matter alterations have reportedly been linked to headache frequency measurements in both episodic and chronic migraine sufferers [START_REF] Neeb | Structural Gray Matter Alterations in Chronic Migraine: Implications for a Progressive Disease?[END_REF]. It is unknown what causes these anatomical alterations in the migraine brain. Some changes might be brought on by a genetic predisposition to migraine attacks.

Repeated occurrences of head pain may lead to other alterations. Such anatomical changes may be the result of brain plasticity, which is the brain's capacity to alter its structure and function in response to changes in the body or the surrounding environment. A variety of CNS modifications, including angiogenesis, synaptogenesis, gliogenesis, neurogenesis, enhancement in cell size, increase in myelin size, and augmentation in blood flow or interstitial fluid, can lead to changes in gray matter.

Axonal remodeling and variations in blood flow are typically the causes of alterations in white matter [START_REF] May | Experience-Dependent Structural Plasticity in the Adult Human Brain[END_REF]. Although there have been several reports of structural alterations in migraine sufferers' brains, it is still unclear how important these changes are to the biology of migraine. However, the presence of structural modifications suggests that migraine causes a progressive anatomical transformation in the brain, which may play an evolving role in the development of the condition and any accompanying disability.

VI. Migraine Treatments

The treatment of migraine aims at limiting the triggers, finding the appropriate abortive treatment, and applying an effective long-term preventive treatment (S. D. [START_REF] Silberstein | Evidence-Based Guideline Update: Pharmacologic Treatment for Episodic Migraine Prevention in Adults: Report of the Quality Standards Subcommittee of the American Academy of Neurology and the American Headache Society[END_REF]. Pharmacological treatments are divided into two categories:

abortive treatments which must be taken at the onset of the migraine attack to be effective, and preventive treatments whose objective is to reduce the frequency and intensity of attacks. Non-pharmacological approaches include behavioral therapy and biofeedback, education, relaxation, mindfulness, and diet adaptation among others.

Abortive Drugs

The goals of acute migraine treatment (with or without aura) are to obtain freedom of pain 2 h after medication intake (significant pain relief is also acceptable)

with 24 h sustained response and without (or with minimal) adverse events. According to the revised guidelines of the French Headache Society [START_REF] Ducros | Revised Guidelines of the French Headache Society for the Diagnosis and Management of Migraine in Adults. Part 2: Pharmacological Treatment[END_REF]) abortive drugs can be classed into two categories:

▪ Non-specific abortive drugs include the use of non-steroidal anti-inflammatory drugs (NSAIDs: diclofenac, ibuprofen, ketoprofen, naproxen) and analgesic drugs (paracetamol, aspirin). They are the cyclooxygenase (COX) inhibitors currently used in the management and treatment of pain, with analgesic, anti-inflammatory, and antipyretic properties. Evidence shows that aspirin (acetylsalicylic acid) with or without metoclopramide (an anti-emetic drug), and most NSAIDs are effective acute migraine treatments [START_REF] Kirthi | Aspirin with or without an Antiemetic for Acute Migraine Headaches in Adults[END_REF]. Paracetamol (acetaminophen), alone or in combination with codeine, caffeine, or aspirin is also effective in reducing migraine pain, but only in attacks of mild-to-moderate intensity with few bothersome symptoms [START_REF] Lipton | Efficacy and Safety of Acetaminophen in the Treatment of Migraine: Results of a Randomized, Double-Blind, Placebo-Controlled, Population-Based Study[END_REF].

▪ Specific abortive drugs include triptans and gepants (see below).

Opioids are not recommended to treat migraine attacks as they exacerbate nausea and increase the risk of MOH. Ditans are serotonin receptor agonists like triptans but with specificity for the subtype 1F (5-HT1F). These receptors are very little present in the periphery and almost absent at the vascular level, but they are found in abundance in the CNS (Vila-Pueyo 2018). The only ditan currently being used to treat acute migraine attacks is lasmiditan [START_REF] Lamb | Fremanezumab in the Prevention of Migraine: A Profile of Its Use[END_REF], which is primarily used in cases of patients with acute migraine and cardiovascular risks.

Triptans

Triptans are recommended as first-line drugs for patients suffering from moderate to severe migraine, associated with disability, who do not respond to nonspecific abortive drugs. They are agonists of serotonin 1B and 1D (5-HT1B/D) receptors, with a lower affinity for other serotonergic receptors, such as 1A, 1E, and 1F. Seven triptans are available in France: almotriptan, eletriptan, frovatriptan, naratriptan, rizatriptan, sumatriptan , and zolmitriptan [START_REF] Ducros | Revised Guidelines of the French Headache Society for the Diagnosis and Management of Migraine in Adults. Part 2: Pharmacological Treatment[END_REF]. Sumatriptan was the first to be marketed, at the beginning of the 1990s. Even if it is absorbed orally at a fast rate, its bioavailability is only 14% and it has a short half-life of about 2 h. The other six triptans were later introduced, which have greater oral bioavailability, longer plasma half-life, active metabolites, higher lipophilicity, and greater potency and affinity for 5-HT1B/D receptors (P. [START_REF] Tfelt-Hansen | Triptans in Migraine: A Comparative Review of Pharmacology, Pharmacokinetics and Efficacy[END_REF].

Clinical trials show that triptans have superior or equal effectiveness at relieving acute migraine pain to NSAIDs and paracetamol [START_REF] Cameron | Triptans in the Acute Treatment of Migraine: A Systematic Review and Network Meta-Analysis[END_REF]. A multiple treatment comparison meta-analysis done in 2013 ranked eletriptan as the most effective triptan at relieving pain at 2 h and 24 h. Rizatriptan was the second most effective but did not have the same efficacy at 24 h, followed by sumatriptan [START_REF] Thorlund | Comparative Efficacy of Triptans for the Abortive Treatment of Migraine: A Multiple Treatment Comparison Meta-Analysis[END_REF].

The administration route and dose can also modify the effectiveness of triptans. For example, subcutaneous administration of sumatriptan (6 mg) shows great efficacy in terms of complete pain relief at 2 h, although adverse events were also high [START_REF] Derry | Sumatriptan (All Routes of Administration) for Acute Migraine Attacks in Adults-Overview of Cochrane Reviews[END_REF].

Mechanisms of Action

Triptans inhibit the release of vasoactive and pro-inflammatory neuropeptides (including CGRP), having mainly a vasoconstrictive effect. Studies have also shown an action on trigeminal transmission, by acting on 5-HT1D presynaptic receptors [START_REF] Ho | Randomized Controlled Trial of the CGRP Receptor Antagonist Telcagepant for Migraine Prevention[END_REF]). Sumatriptan, one of the most notable triptans uses its vasoconstrictive action, and hence its ability to reduce the release of CGRP from nociceptive fiber terminals of the trigeminal nerve, and to modulate 5-HT synthesis at the cortical level [START_REF] Humphrey | The Mode of Action of Sumatriptan Is Vascular? A Debate[END_REF]Sakai et al. 2008). The majority of 5-HT1B/D receptors are localized at axon terminals in the presynaptic region of the Sp5c and trigeminal ganglion [START_REF] Ma | Co-Localization of 5-HT1B/1D/1F Receptors and Glutamate in Trigeminal Ganglia in Rats[END_REF]. It is also important to note that 5HT receptors also have a central effect and reside at the central level of other migraine-related regions. The LC, hippocampus, and cortex, all contain low densities of central 5-HT1D receptors with similar distributions among humans and rodents (Tricklebank and Daly 2019). While with respect to 5-HT1B receptors, in humans, high densities have been found in the substantia nigra and globus pallidus [START_REF] Bonaventure | Autoradiographic Mapping of 5-HT1B-and 5-HT1D Receptors in Human Brain Using [3H] Alniditan, a New Radioligand[END_REF][START_REF] Varnäs | Autoradiographic Mapping of 5-HT1B and 5-HT1D Receptors in the Post Mortem Human Brain Using [3H] GR 125743[END_REF]. The dorsal raphe nucleus and the cortex show intermediate densities and lower expressions are in the hippocampus, amygdala, and thalamus [START_REF] Varnäs | Autoradiographic Mapping of 5-HT1B and 5-HT1D Receptors in the Post Mortem Human Brain Using [3H] GR 125743[END_REF][START_REF] Varnäs | Autoradiographic Distribution of Serotonin Transporters and Receptor Subtypes in Human Brain[END_REF]. This distribution is homologous with the distribution in rodents [START_REF] Bruinvels | Autoradiographic Characterisation and Localisation of 5-HT 1D Compared to 5-HT 1B Binding Sites in Rat Brain[END_REF].

Triptan Non-responders

Triptans are highly effective if taken early within the first few minutes of the start of the headache attack. When the attack is established, and there is the presence of cutaneous cephalic hypersensitivity (indicative of ongoing central sensitization) patients are no longer responsive to triptans (Rami Burstein, Collins, and Jakubowski 2004).

Although triptans are recommended as a first-line treatment, approximately 30-40 % of migraineurs suffer from insufficient efficacy or tolerability [START_REF] Lipton | Impact of NSAID and Triptan Use on Developing Chronic Migraine: Results From the American Migraine Prevalence and Prevention (AMPP) Study[END_REF]Viana et al., 2013). Migraine patients can be divided into triptan responders or non-responders.

In the first category of patients, triptan is effective against an acute attack in at least three out of four migraine attacks [START_REF] Sacco | European Headache Federation Guideline on the Use of Monoclonal Antibodies Targeting the Calcitonin Gene Related Peptide Pathway for Migraine Prevention -2022 Update[END_REF], while in the second group of patients, triptan fails to abort the headache. However, triptan non-responders are within a larger group of triptan failures that further includes; triptan resistant (failure of at least 2 different triptans); triptan refractory (failure of at least 3 different triptans, including subcutaneous formulation); and triptan ineligibility (presence of a contraindication) [START_REF] Sacco | European Headache Federation (EHF) Consensus on the Definition of Effective Treatment of a Migraine Attack and of Triptan Failure[END_REF]). In the case of sumatriptan, for example, non-responders show lower and slower absorption after oral administrations compared to responders (A. [START_REF] Ferrari | Interindividual Variability of Oral Sumatriptan Pharmacokinetics and of Clinical Response in Migraine Patients[END_REF]. Moreover, in women, the efficacy rate of triptans is the same as in men, however, their risk of a headache recurrence is higher [START_REF] Casteren | Sex Differences in Response to Triptans: A Systematic Review and Meta-Analysis[END_REF]. It is important also to add that inadequate efficacy and/or tolerability with one triptan does not always predict outcomes with another triptan and switching to another triptan might benefit patients (Viana et al. 2013).

Gepants

Gepants were the primary oral agents designed for both migraine treatment and prevention. They are CGRP receptor antagonists, and include rimegepant, ubrogepant, and olcegepant [START_REF] Croop | Oral Rimegepant for Preventive Treatment of Migraine: A Phase 2/3, Randomised, Double-Blind, Placebo-Controlled Trial[END_REF][START_REF] Dodick | Ubrogepant, an Acute Treatment for Migraine, Improved Patient-Reported Functional Disability and Satisfaction in 2 Single-Attack Phase 3 Randomized Trials, ACHIEVE I and II[END_REF][START_REF] Recober | Olcegepant, a Non-Peptide CGRP1 Antagonist for Migraine Treatment[END_REF]. The functionality of gepants is through their binding to CGRP receptors and inhibiting their activation. They hence block the amplification and progression of the pain process from the trigeminal ganglion, trigeminal nerve fibers and, dura. Ubrogepant, an FDAapproved gepant used for the acute treatment of migraine has shown a highly selective efficacy and is considered a first class of drugs in the class of oral CGRP antagonists [START_REF] Scott | Ubrogepant: First Approval[END_REF]. Gepants are a reasonable alternative for migraineurs that are triptan nonresponders and for those with triptan contraindications (Moreno-Ajona, Villar-Martínez, and Goadsby 2022). For instance, the efficacy and tolerability of ubrogepant showed no difference compared to that of groups using triptans [START_REF] Blumenfeld | Efficacy of Ubrogepant Based on Prior Exposure and Response to Triptans: A Post Hoc Analysis[END_REF].

Moreover, the coadministration of ubrogepant with other triptans such as sumatriptan is well tolerated by patients with minimal side effects [START_REF] Jakate | Evaluation of the Pharmacokinetic Interaction of Ubrogepant Coadministered With Sumatriptan and of the Safety of Ubrogepant With Triptans[END_REF]).

Prophylactic Treatments

Preventive migraine treatments aim at reducing monthly migraine days by at least 50% in episodic migraine and by at least 30% in chronic migraine. Prophylaxis also aims at reducing the consumption of acute treatments, intensity and duration of attacks, and improving the quality of life [START_REF] Ducros | Revised Guidelines of the French Headache Society for the Diagnosis and Management of Migraine in Adults. Part 2: Pharmacological Treatment[END_REF]. Most patients under prophylaxis will still have attacks, which will be treated by abortive approaches. In Europe, commonly prescribed prophylactic agents include:

▪ Anticonvulsant drugs (venlafaxine, valproate, and topiramate). Their effect is exerted by the inhibition of calcium channels that limit the release of excitatory molecules or increase the release of GABA (γ-Aminobutyric acid)(Mattias [START_REF] Linde | Topiramate for the Prophylaxis of Episodic Migraine in Adults[END_REF]. Topiramate is the main potent anticonvulsant drug against episodic migraine with high efficacy (Stephen D. Silberstein 2015).

▪ Antihypertensives, block both β1 and β2 receptor subtypes, and therefore, the action of the endogenous catecholamines, adrenaline, and noradrenaline (NA).

Propranolol is the most common and one of the most effective first-line medications used for migraine prophylaxis, which presents high effectiveness in cases of episodic migraine (J. L. [START_REF] Jackson | Beta-Blockers for the Prevention of Headache in Adults, a Systematic Review and Meta-Analysis[END_REF]. Other β-blockers that can be used are timolol, atenolol, and metoprolol, which should be considered in patients with underlying cardiovascular diseases.

▪ Antidepressant. Evidence shows that amitriptyline, a tricyclic antidepressant, is superior to a placebo to reduce headaches by 50% in episodic migraine, with an efficacy comparable to that of propranolol [START_REF] Ziegler | Migraine Prophylaxis: A Comparison of Propranolol and Amitriptyline[END_REF]. A systematic review concluded that venlafaxine, a dual reuptake inhibitor, had antimigraine effectiveness (F. Wang et al. 2020) while no evidence supports the prophylactic effect of selective serotonin reuptake inhibitors.

▪ Onabotulinumtoxin A (Botox) has been pivotal in chronic migraine through its efficacy in reducing monthly headache days and improving the quality of life in addition to being well tolerated by patients [START_REF] Escher | Botulinum Toxin in the Management of Chronic Migraine: Clinical Evidence and Experience[END_REF]). However, it has not shown any superiority over the placebo in cases of episodic migraine [START_REF] Herd | Botulinum Toxins for the Prevention of Migraine in Adults[END_REF].

▪ Monoclonal anti-CGRP molecules, targeting CGRP or its receptor, represent the first specific and selective migraine prophylactic treatment [START_REF] Garland | Erenumab: A First-in-Class Monoclonal Antibody for Migraine Prevention[END_REF]. Anti-CGRP antibodies are effective and well tolerated in patients with chronic migraine even in the presence of prior failed therapies or medication overuse [START_REF] Caronna | Anti-CGRP Monoclonal Antibodies in Chronic Migraine with Medication Overuse: Real-Life Effectiveness and Predictors of Response at 6 Months[END_REF]. In one recent study, eptinezumab, erenumab, fremanezumab, and galcanezumab significantly reduced the monthly migraine days compared to placebo (X. [START_REF] Wang | Efficacy and Safety of Monoclonal Antibody Against Calcitonin Gene-Related Peptide or Its Receptor for Migraine: A Systematic Review and Network Meta-Analysis[END_REF]). However, safety-wise, galcanezumab solely increased the incidences of treatment-emergent adverse events.

Non-pharmacological Interventions

Changing one's daily dietary intake and designing a systematic, and mechanismdriven diet where dietary supplements are introduced such as vitamin B2 and food rich in magnesium and omega-3 can provide both medicinal and health benefits for migraine [START_REF] Puledda | Non-Pharmacological Approaches for Migraine[END_REF]. In addition, the usage of behavioral techniques (such as cognitive behavioral therapy, and relaxation techniques), acupuncture, and hypnosis have aimed at helping patients to further cope with their symptoms. They have evidence of positive functionality to a certain degree in migraineurs where the treatments are no longer functional [START_REF] Penzien | Well-Established and Empirically Supported Behavioral Treatments for Migraine[END_REF]. Applying changes to one's lifestyle can also improve the effectiveness of the treatment, the usage of stress reduction methods and the introduction of physical activity can enhance treatment efficacy and one's quality of life [START_REF] Wells | Effectiveness of Mindfulness Meditation vs Headache Education for Adults With Migraine[END_REF].

Non-invasive neuromodulation is also used by utilizing techniques that stimulate the central and peripheral nervous systems through either an electric current or magnetic field through the skin such as transcutaneous cranial nerve stimulation [START_REF] Schoenen | Migraine Prevention with a Supraorbital Transcutaneous Stimulator: A Randomized Controlled Trial[END_REF]). This field is backed up by evidence of functionality in migraine however it is only located in specialized centers [START_REF] Puledda | Non-Pharmacological Approaches for Migraine[END_REF]. Moreover, invasive neuromodulation techniques also exist such as occipital nerve stimulation but those are limited to the highest cases of refractory migraineurs with multiple failed interventions [START_REF] Bartsch | Stimulation of the Greater Occipital Nerve Induces Increased Central Excitability of Dural Afferent Input[END_REF].

VII. Animal Models of Migraine

Various animal models have been designed to understand the pathophysiology of migraine. So far there has not been an animal model that can replicate all the features of this complex disorder. Currently, the focus of animal model designs is to stimulate the TCC. Dura mater, large cerebral arteries, and meningeal vasculature are painsensitive areas innervated by nociceptors located on meningeal and trigeminovascular afferents derived from the trigeminal nerve. Stimulation of the nerves innervating these structures leads to the induction of headaches similar to migraine [START_REF] Olesen | Origin of Pain in Migraine: Evidence for Peripheral Sensitisation[END_REF]).

The most investigated animal models are the ones based on chemical stimulations using different vasodilating agents. The "inflammatory soup" model using a mix of inflammatory mediators [prostaglandin E2 (PGE-2), histamine, 5-HT, and bradykinin] administered through a micro-catheter at the level of the meninges is used for meningeal and trigeminovascular stimulation hence inducing cephalic mechanical sensitivity (Boyer et al. 2014;[START_REF] Munro | Animal Models of Pain and Migraine in Drug Discovery[END_REF]. One of the limitations of this technique is that the usage of such chemicals can compromise the functionality of the BBB.

Other models use systemic administration of NO donors, such as NTG or ISDN.

Typically, NTG infusion induces headache (often with premonitory symptoms) in up to 83% of patients with migraine without aura, and in 67% of those with migraine with aura [START_REF] Goadsby | A Review of Paroxysmal Hemicranias, SUNCT Syndrome and Other Short-Lasting Headaches with Autonomic Feature, Including New Cases[END_REF]. Attacks are not provoked in controls who do not have migraine and NTG does not induce aura. In mice, NTG induces mechanical and thermal extracephalic hypersensitivity which are symptoms reversed by sumatriptan [START_REF] Bates | Sumatriptan Alleviates Nitroglycerin-Induced Mechanical and Thermal Allodynia in Mice[END_REF]). In addition, NTG-injected mice experience light aversion (as a surrogate of clinical photophobia) and augmentation of meningeal blood flow (K. C. [START_REF] Brennan | Casein Kinase Iδ Mutations in Familial Migraine and Advanced Sleep Phase[END_REF]Markovics et al. 2012). Intermittent injections of NTG were also used to assess the progression of migraine from an acute to a chronic state, in which extracephalic mechanical hypersensitivity became progressive and sustained [START_REF] Pradhan | Characterization of a Novel Model of Chronic Migraine[END_REF].

Even with its positive aspects, this model has some limitations. First, NTG is hypotensive and requires a vehicle consisting of alcohol and propylene glycol hence having non-specific effects [START_REF] Ramachandran | A Naturalistic Glyceryl Trinitrate Infusion Migraine Model in the Rat[END_REF]. Second, repetitive administrations of NTG induce anxiety in rats [START_REF] Taheri | Nitric Oxide Role in Anxiety-like Behavior, Memory and Cognitive Impairments in Animal Model of Chronic Migraine[END_REF]) which could limit the study of light avoidance as a migraine-associated symptom. Finally, in most of the studies using NTG in rodents there were no differences between males and females (Targowska-Duda et al. 2020), and the data were often pooled [START_REF] Jalgaonkar | Analgesic, Anti-Inflammatory, and Antioxidant Potential of S-Adenosyl L-Methionine on Nitroglycerine Induced Migraine in Mice Models[END_REF].

ISDN has been already validated to trigger headaches in humans (Bellantonio et al. 1997;Castellano et al. 1998). In male rats, a single systemic ISDN injection induces reversible cephalic mechanical hypersensitivity that can be aborted by sumatriptan and olcegepant (Dallel, Descheemaeker, and Luccarini 2018). Furthermore, repeated ISDN administrations provoke persistent cephalic and transitory extracephalic mechanical hypersensitivities, both of which are prevented by propranolol (Dallel, Descheemaeker, and Luccarini 2018). ISDN also induces central sensitization of trigeminal pathways (Flores Ramos et al. 2017) that is correlated to migraine progression into chronic forms.

Compared to NTG, ISDN has its pros, starting with being less hypotensive [START_REF] Manabe | Tolerance to Nitroglycerin Induced by Isosorbide-5-Mononitrate Infusion in Vivo[END_REF]). Moreover, it comes in an injectable form hence being easily administered to both humans and animals allowing for a mimic of what occurs in clinical settings. Last but not least, it does not require a vehicle such as alcohol or propylene glycol to be dissolved in, hence this removes the non-specific variables [START_REF] Ramachandran | A Naturalistic Glyceryl Trinitrate Infusion Migraine Model in the Rat[END_REF].

A similar migraine provocation pattern as with NTG has been seen with CGRP.

In humans, an intravenous administration of CGRP is capable of inducing headaches [START_REF] Asghar | Dilation by CGRP of Middle Meningeal Artery and Reversal by Sumatriptan in Normal Volunteers[END_REF]. When it comes to animals, subcutaneous injections of CGRP in male rodents induced periorbital mechanical hypersensitivity [START_REF] Logu | Migraine-Provoking Substances Evoke Periorbital Allodynia in Mice[END_REF]). In addition, dural administrations of CGRP, induced periorbital touch hypersensitivity in mice [START_REF] Avona | Dural Calcitonin Gene-Related Peptide Produces Female-Specific Responses in Rodent Migraine Models[END_REF].

CSD, a slow wave of depolarization of neuronal and glial cells in the cortex, forms another migraine model that can be induced by injections of potassium chloride (KCl) [START_REF] Andreou | Animal Models of Headache: From Bedside to Bench and Back to Bedside[END_REF]. It can also be induced by electrical and mechanical stimulations of the cortex in rodents [START_REF] Eikermann-Haerter | Animal Models of Migraine Headache and Aura[END_REF]. This technique does remain invasive and behavioral assessments can't be done since the animal is anesthetized.

VIII. The Noradrenergic System

Synthesis, Release, and Reuptake of Noradrenaline

NA is a monoamine that, like adrenaline and dopamine, belongs to the catecholamine group and is synthesized from the amino acid L-tyrosine. Each stage in the synthesis of NA (Figure 8) is dependent on a distinct enzyme that acts as a catalyst, with the rate-limiting enzyme being TH.

NA is deposited in synaptic vesicles, and released into the synaptic cleft following an electrical input to the noradrenergic terminal. The release is regulated by a negative feedback that involves the presynaptic α2adrenoreceptors (AR) [START_REF] Bukharaeva | Adrenoceptors Modulate Cholinergic Synaptic Transmission at the Neuromuscular Junction[END_REF]. NA then binds to presynaptic and postsynaptic AR and the signal is either propagated or inhibited depending on the AR subtype. NA can either be taken up by the NA transporter (NAT) or degraded by the monoamine oxidase (MAO) A and catechol-O-methyltransferase, enzymes located within the synaptic cleft and nerve terminal.

The Noradrenergic Regions in the CNS

In rodents and primates, the noradrenergic system is primarily composed of seven separate clusters of cell bodies that are located in the brainstem. The pontine's lateral and ventral reticular formations, respectively, are home to the A1 and A7 cell groups. The nucleus of the solitary tract contains A2 neurons. A3 and A4 are located in the roof of the fourth ventricle, ventral to the cerebellar nuclei. A5 is located close to the superior olivary complex in the pontine tegmentum while the LC (A6) is located in the dorsal wall of the rostral pons on the lateral floor of the fourth ventricle [START_REF] Dahlström | Localization of Monoamines in the Lower Brain Stem[END_REF][START_REF] Felten | Monoamine Distribution in Primate Brain V. Monoaminergic Nuclei: Anatomy, Pathways and Local Organization[END_REF]. These noradrenergic areas send axonal projections to different nuclei of the CNS, but the target regions from each nucleus differ considerably.

The Locus Coeruleus

The noradrenergic LC has been implicated in a variety of physiological and neurobiological functions such as cardiovascular regulation, arousal, vigilance, decision-making, attention, learning, memory, sleep-wake cycle, motor activity, and cognitive activation [START_REF] Clayton | Phasic Activation of Monkey Locus Ceruleus Neurons by Simple Decisions in a Forced-Choice Task[END_REF][START_REF] James | Locus Coeruleus in Memory Formation and Alzheimer's Disease[END_REF][START_REF] Maness | Role of the Locus Coeruleus and Basal Forebrain in Arousal and Attention[END_REF]).

Moreover, it has been also established to be intimately related to the etiology of neuropathological and psychiatric states, for example, stress, Alzheimer's disease, depression, and anxiety disorders [START_REF] Giorgi | The Connections of Locus Coeruleus with Hypothalamus: Potential Involvement in Alzheimer's Disease[END_REF][START_REF] Itoi | The Brainstem Noradrenergic Systems in Stress, Anxiety and Depression[END_REF]. The involvement of the LC in pain modulation and specifically in migraine will be detailed below.

Anatomical and Molecular Organization

The LC is considered the principal noradrenergic source in the CNS [START_REF] Dahlstroem | Evidence for the Existence of Monoamine Neurons in the Central Nervous System. I. Demonstration of Monoarnines in Cell Badies of Brainstem Neurons[END_REF] in all mammalian species (Russell 1955), with an elaborate network of ascending and descending projections. It consists of two bilateral nuclei located on either side of the dorsal wall of the rostral pons on the lateral floor of the fourth ventricle [START_REF] Amaral | The Locus Coeruleus: Neurobiology of a Central Noradrenergic Nucleus[END_REF]. Its size is reduced with an average diameter of ~300 μm in mice, and only ∼1,500 neurons in mice against 20,000 in humans in each hemisphere [START_REF] Sara | Orienting and Reorienting: The Locus Coeruleus Mediates Cognition through Arousal[END_REF][START_REF] Schwarz | Organization of the Locus Coeruleus-Norepinephrine System[END_REF]. The cluster of NA neurons located in the LC constitutes about half of the noradrenergic neurons of the CNS. Studies have shown that most LC neurons synthesize the enzyme dopamine βhydroxylase which is responsible for the conversion of dopamine to NA hence deducing that NA is the primary neurotransmitter released [START_REF] Grzanna | The Locus Coeruleus in the Rat: An Immunohistochemical Delineation[END_REF][START_REF] Mateo | Somatodendritic Α2-Adrenoceptors in the Locus Coeruleus Are Involved in the In Vivo Modulation of Cortical Noradrenaline Release by the Antidepressant Desipramine[END_REF]. In addition to NA, other neuropeptides and neuromodulators are expressed and released by the LC, such as galanin, which is expressed in up to 80%

of neurons and plays a role in wake/sleep states, and nociception, NPY, vasopressin, somatostatin, and enkephalin [START_REF] Holets | Locus Coeruleus Neurons in the Rat Containing Neuropeptide Y, Tyrosine Hydroxylase or Galanin and Their Efferent Projections to the Spinal Cord, Cerebral Cortex and Hypothalamus[END_REF][START_REF] Lang | Physiology, Signaling, and Pharmacology of Galanin Peptides and Receptors: Three Decades of Emerging Diversity[END_REF][START_REF] Sutin | Chapter 1 -Neurochemicals in the Dorsal Pontine Tegmentum[END_REF][START_REF] Tanaka | Met-Enkephalin, Injected during the Early Phase of Stress, Attenuates Stress-Induced Increases in Noradrenaline Release in Rat Brain Regions[END_REF]. Moreover, recent studies showed a GABAergic population of neurons that could modulate the activity of noradrenergic neurons (Breton-Provencher & Sur, 2019). rostral, middle, or caudal according to the mouse brain atlas. The rostral section starts from AP -5.2 to AP -5.34, relative to Bregma, followed by the middle portion (from AP -5.34 to AP -5.52) and finished in the most caudal slide (from AP -5.52 to AP -5.8 me5: mesencephalic trigeminal nucleus; LC: locus coeruleus. Edited and Adapted from (Paxinos & Franklin, 2019).

Noradrenergic cells in the LC exhibit different well-organized morphologies with rostrocaudal (Figure 9) and dorsoventral orientations (Figure 10). At least two subpopulations have been described: large multipolar cells (approx. 35 μm) located more ventrally, and small fusiform cells (approx. 20 μm) located more dorsally in rats [START_REF] Grzanna | The Locus Coeruleus in the Rat: An Immunohistochemical Delineation[END_REF][START_REF] Swanson | The Locus Coeruleus: A Cytoarchitectonic, Golgi and Immunohistochemical Study in the Albino Rat[END_REF]). There also exists a third group of scattered noradrenergic neurons not specified in terms of structure. Noradrenergic neurons can be also described according to their molecular composition, including numerous AR subtypes, with α2 being the most prevalent and α1 being less prevalent (W. S. [START_REF] Young | Noradrenergic Alpha 1 and Alpha 2 Receptors: Light Microscopic Autoradiographic Localization[END_REF]. α2-ARs are heavily located at the level of the posterior LC while α1-ARs are more abundant in the anterior LC [START_REF] Chamba | Distribution of Alpha-1 and Alpha-2 Binding Sites in the Rat Locus Coeruleus[END_REF]. Neurons express also nicotinic ACh receptors, GABA, orexin/hypocretin, and opioid receptors [START_REF] Luque | Localization of GABAA Receptor Subunit MRNAs in the Rat Locus Coeruleus[END_REF][START_REF] Mansour | μ-Opioid Receptor MRNA Expression in the Rat CNS: Comparison to μ-Receptor Binding[END_REF][START_REF] Marcus | Differential Expression of Orexin Receptors 1 and 2 in the Rat Brain[END_REF].

Afferent and Efferent Networks

Through retrograde and anterograde tracing studies, the LC was shown to be composed of various specialized noradrenergic modules each comprising distinct populations of NA neurons with wide projection targets and receiving inputs from a diverse array of brain regions (Figure 11A) [START_REF] Totah | Noradrenergic Ensemble-Based Modulation of Cognition over Multiple Timescales[END_REF][START_REF] Uematsu | Modular Organization of the Brainstem Noradrenaline System Coordinates Opposing Learning States[END_REF].

At the level of the brainstem and midbrain, the LC receives projections of a nociceptive nature from PAG, reticular formation, nucleus tractus solitarius, and vestibular nucleus, in addition to information about both the visceral and sympathetic nervous system (Aston- [START_REF] Jones | Quantitative Characterization of Ceruleospinal Inhibition of Nociceptive Transmission in the Rat[END_REF][START_REF] Van Bockstaele | Light and Electron Microscopic Evidence for Topographic and Monosynaptic Projections from Neurons in the Ventral Medulla to Noradrenergic Dendrites in the Rat Locus Coeruleus[END_REF]. The LC is also a recipient of complex emotional, cognitive, and homeostatic information from the hypothalamus (dorsomedial, lateral, and paraventricular nuclei), prefrontal cortex, insular cortex, and the bed nucleus of the stria terminalis [START_REF] Luppi | Afferent Projections to the Rat Locus Coeruleus Demonstrated by Retrograde and Anterograde Tracing with Cholera-Toxin B Subunit and Phaseolus Vulgaris Leucoagglutinin[END_REF][START_REF] Reyes | Hypothalamic Projections to Locus Coeruleus Neurons in Rat Brain[END_REF][START_REF] Van Bockstaele | Light and Electron Microscopic Evidence for Topographic and Monosynaptic Projections from Neurons in the Ventral Medulla to Noradrenergic Dendrites in the Rat Locus Coeruleus[END_REF]. Other afferent connections are between the LC and other neuromodulatory brain regions such as the serotoninergic dorsal raphe nucleus and the dopaminergic ventral tegmental area [START_REF] Deutch | Activation of the Locus Coeruleus Induced by Selective Stimulation of the Ventral Tegmental Area[END_REF][START_REF] Ornstein | Biochemical and Radioautographic Evidence for Dopaminergic Afferents of the Locus Coeruleus Originating in the Ventral Tegmental Area[END_REF]. The aforementioned afferent connections, pave the way for the LC's neural processing and regulation of sensory and visceral experiences, through the conveyance of highly processed cognitive and emotional information from higher-order structures.

The LC is a broad projector toward most forebrain regions in addition to the cerebellum, spinal cord, and some midbrain and brainstem nuclei (Aston- [START_REF] Aston-Jones | Adaptive Gain and the Role of the Locus Coeruleus-Norepinephrine System in Optimal Performance[END_REF][START_REF] Berridge | The Locus Coeruleus-Noradrenergic System: Modulation of Behavioral State and State-Dependent Cognitive Processes[END_REF][START_REF] Valentino | Endogenous Opioids: The Downside of Opposing Stress[END_REF]. It sends projections related to learning and memory toward the amygdala and the medial prefrontal cortex (Amy F. T. [START_REF] Arnsten | Stress Signalling Pathways That Impair Prefrontal Cortex Structure and Function[END_REF]. The heterogeneous specific nature of the LC neurons in terms of their efferent connectivity was assessed using a combinatorial strategy using two or more retrograde tracers. Interestingly, Chandler and Waterhouse were able to detect three non-overlapping cell populations after injecting different retrograde tracers at the level of the medial prefrontal cortex, orbitofrontal cortex, and anterior cingulate cortex (Figure 11B) (D. Chandler and Waterhouse 2012). The same team was also able to find another population, distinct from the others already found with a lower excitatory rate, projecting at the level of the motor cortex (D. J. [START_REF] Chandler | Heterogeneous Organization of the Locus Coeruleus Projections to Prefrontal and Motor Cortices[END_REF]. It has been also mentioned that the efferent connectivity of the LC may be governed by some form of functional demand. LC neurons have been shown to send more prominent axon collaterals toward the somatosensory cortex while less toward the visual cortical/thalamic brain regions [START_REF] Simpson | Lateralization and Functional Organization of the Locus Coeruleus Projection to the Trigeminal Somatosensory Pathway in Rat[END_REF]. All of this adds to the idea that the functional demand could be the underlying player in the degree of connectivity and collateralization exhibited by sole populations of LC neurons. In addition, there exists a topographical connection between the LC according to its rostrocaudal orientation within the other areas of the CNS. Within the caudal LC, noradrenergic neurons project toward the thalamus, cortex, and amygdala, while the rostral LC projects to the hippocampus, septum, and hypothalamus mainly. As for the middle portion of the LC it sends its projections towards the hippocampus, cerebellum, and spinal cord [START_REF] Schwarz | Organization of the Locus Coeruleus-Norepinephrine System[END_REF]. The LC also sends NA projections toward the and β-AR (β1, β2, β3), which stimulate Gs proteins in the postsynaptic membrane [START_REF] Bylund | International Union of Pharmacology Nomenclature of Adrenoceptors[END_REF]). In the LC, α2-ARs are primarily α2A and α2C and are located preand postsynaptically in noradrenergic neurons or presynaptically on non-noradrenergic terminals [START_REF] Bücheler | Two Α2-Adrenergic Receptor Subtypes, Α2A and Α2C, Inhibit Transmitter Release in the Brain of Gene-Targeted Mice[END_REF][START_REF] Gyires | Α2-Adrenoceptor Subtypes-Mediated Physiological, Pharmacological Actions[END_REF]).

The main function of presynaptic α2-AR is acting as an autoinhibitory feedback to control the NA release from the neuron itself and terminal areas [START_REF] Starke | Presynaptic Autoreceptors in the Third Decade: Focus on A2-Adrenoceptors[END_REF]. The NA binding to α2-AR triggers the activation of a Gi/o protein-coupled cascade:

inhibition of adenylyl cyclase and reduction of cAMP levels, a decrease of Ca 2+ channel opening, activation of K + channels, and phosphorylation of the mitogenactivated protein kinase (MAPK) pathway [START_REF] Gilsbach | Presynaptic Metabotropic Receptors for Acetylcholine and Adrenaline/Noradrenaline[END_REF]. These mechanisms modulated by regulators of G-protein signaling, result in presynaptic NA release inhibition.

As described above, the LC receives numerous innervations from the CNS and even, there is evidence of local non-adrenergic circuits. Several studies have described the presence of GABA, N-methyl-D-aspartate (NMDA), nicotinic, purine, and corticotropin-releasing factor receptors in the LC (Breton-Provencher and Sur 2019; [START_REF] Karolewicz | Elevated Levels of the NR2C Subunit of the NMDA Receptor in the Locus Coeruleus in Depression[END_REF][START_REF] Léna | Diversity and Distribution of Nicotinic Acetylcholine Receptors in the Locus Ceruleus Neurons[END_REF][START_REF] Wang | Corticotropin-Releasing Factor Receptors in the Locus Coeruleus Modulate the Enhancement of Active Coping Behaviors Induced by Chronic Predator Odor Inoculation in Mice[END_REF][START_REF] Yao | Purinergic Modulation of Cardiovascular Function in the Rat Locus Coeruleus[END_REF]. For example, glutamatergic responses of LC neurons are mediated largely via ionotropic receptors (NMDA receptors) present in noradrenergic neurons, while GABA responses are mediated by GABAA receptors [START_REF] Chandley | Elevated Gene Expression of Glutamate Receptors in Noradrenergic Neurons from the Locus Coeruleus in Major Depression[END_REF][START_REF] Koga | Activation of Presynaptic GABAA Receptors Increases Spontaneous Glutamate Release onto Noradrenergic Neurons of the Rat Locus Coeruleus[END_REF]).

The serotoninergic system also plays a role in the functionality of the LC. The LC is a recipient of a robust innervation from serotoninergic nuclei, including the dorsal raphe nucleus [START_REF] Pudovkina | The Interaction between the Locus Coeruleus and Dorsal Raphe Nucleus Studied with Dual-Probe Microdialysis[END_REF]. The dorsal raphe nucleus through the activation of the 5-HT1A and 5-HT2A receptors is capable of exerting an effect on the LC [START_REF] Haddjeri | Modulation of the Firing Activity of Noradrenergic Neurones in the Rat Locus Coeruleus by the 5-Hydroxtryptamine System[END_REF]. Reductions in the spontaneous and pain-evoked activity of the LC occur post the stimulation of serotoninergic receptors [START_REF] Segal | Serotonergic Innervation of the Locus Coeruleus from the Dorsal Raphe and Its Action on Responses to Noxious Stimuli[END_REF]. While the application of serotonin in the LC attenuates the sensorial, neurochemical, and electrical evoked excitatory responses of the LC [START_REF] Aston-Jones | Serotonin Selectively Attenuates Glutamate-Evoked Activation of Noradrenergic Locus Coeruleus Neurons[END_REF][START_REF] Bobker | Serotonin Agonists Inhibit Synaptic Potentials in the Rat Locus Ceruleus in Vitro via 5-Hydroxytryptamine1A and 5-Hydroxytryptamine1B Receptors[END_REF]. Moreover, further validating the modulatory role of serotonin, studies have shown that serotonin modulates the release of NA. Interestingly, through a study using in vivo microdialysis in rats, the infusion of the 5-HT1A receptor agonist flesinoxan into the LC decreased the release of NA in this structure [START_REF] Pudovkina | The Release of Noradrenaline in the Locus Coeruleus and Prefrontal Cortex Studied with Dual-Probe Microdialysis[END_REF].

Involvement of the LC in Pain

Numerous studies have also shown that the LC aids in the sensory and emotional integration of pain. Indeed, it is activated by various stimuli (such as tail pinch, foot shock, and heat), which increases the firing rate and NA turnover, and suggests that this nucleus plays a role in pain perception [START_REF] Taylor | The Noradrenergic Locus Coeruleus as a Chronic Pain Generator[END_REF].

Although some studies suggest that LC has pain-facilitating properties, its function has traditionally been understood as a source of pain inhibition [START_REF] Brightwell | Noradrenergic Neurons in the Locus Coeruleus Contribute to Neuropathic Pain[END_REF]. Indeed, descending noradrenergic projections to the spinal cord were described some time ago and characterized as pain inhibitory (S. L. [START_REF] Jones | Quantitative Characterization of Ceruleospinal Inhibition of Nociceptive Transmission in the Rat[END_REF][START_REF] Kwiat | The Origin of Brainstem Noradrenergic and Serotonergic Projections to the Spinal Cord Dorsal Horn in the Rat[END_REF][START_REF] Westlund | Descending Projections of the Locus Coeruleus and Subcoeruleus/Medial Parabrachial Nuclei in Monkey: Axonal Transport Studies and Dopamine-Beta-Hydroxylase Immunocytochemistry[END_REF]. Subsequent studies in awake animals often conclude that tonic activation of LC reduces acute nociception(W. J. [START_REF] Martin | Differential Effects of Neurotoxic Destruction of Descending Noradrenergic Pathways on Acute and Persistent Nociceptive Processing[END_REF]). The LC is also capable to promote the feedback inhibition of the persistent nociception that arises after tissue or nerve injury. For example, molecular genetic silencing of the LC produced cutaneous hypersensitivity and increased the heat hypersensitivity associated with inflammation [START_REF] Howorth | Retrograde Adenoviral Vector Targeting of Nociresponsive Pontospinal Noradrenergic Neurons in the Rat in Vivo[END_REF]. The LC promotes analgesia by activating α2-AR, in particular the subtype 2A, that is expressed on the primary afferents in the spinal cord [START_REF] Hentall | Temporal and Spatial Profiles of Pontine-Evoked Monoamine Release in the Rat's Spinal Cord[END_REF]. Spinal delivery of α2-AR agonists reduces signs of pain in both animals and humans [START_REF] Baba | Norepinephrine Facilitates Inhibitory Transmission in Substantia Gelatinosa of Adult Rat Spinal Cord (Part 1) Effects on Axon Terminals of GABAergic and Glycinergic Neurons[END_REF][START_REF] Kawasaki | Α2Adrenoceptor-Mediated Presynaptic Inhibition of Primary Afferent Glutamatergic Transmission in Rat Substantia Gelatinosa Neurons[END_REF], while intrathecal injection α2-AR antagonist produces hyperalgesia as well as Fos expression in the dorsal horn (Hughes et al. 2013). Gabapentin (an anticonvulsant drug used to treat chronic pain) activates the LC to induce spinal NA release that in turn stimulates α2-AR, leading to analgesia [START_REF] Hayashida | Gabapentin Acts within the Locus Coeruleus to Alleviate Neuropathic Pain[END_REF].

Under certain conditions, intense or long-term sensory stimulation invokes mechanisms that produce brain nociceptive facilitation that can persist long after the injury has healed, contributing to central sensitization that drives the neural and behavioral manifestations of chronic pain. Hence, both descending pain modulatory outflows from the LC to the spinal cord and trigeminal dorsal horns, as well as ascending pain modulatory outflows to the cortex become predominantly facilitatory.

Pronociceptive and antinociceptive actions are mediated by distinct subpopulations of LC neurons: the antinociceptive effect is originated from the ventral region and subcoeruleus that project to the dorsal horn of the spinal cord [START_REF] Bruinstroop | Hypothalamic Neuropeptide Y (NPY) Controls Hepatic VLDL-Triglyceride Secretion in Rats via the Sympathetic Nervous System[END_REF][START_REF] Howorth | Retrograde Adenoviral Vector Targeting of Nociresponsive Pontospinal Noradrenergic Neurons in the Rat in Vivo[END_REF] while pronociceptive effects would originate from neurons localized more dorsally.

In addition to controlling sensory pain, the LC modulates the cortico-thalamic loop, which processes the emotional aspect of pain, by sending ascending signals to cortical regions like the prefrontal cortex (Gary Aston-Jones and Cohen 2005).

Additionally, several findings evidence the pivotal role of the LC on the antalgic effect of antidepressants in chronic pain [START_REF] Seki | Molecular Mechanism of Noradrenaline during the Stress-Induced Major Depressive Disorder[END_REF]. This effect seems to be related to the delay of the onset of the action of antidepressants.

Involvement of the LC in Migraine

Both preclinical and clinical evidence support the potential involvement of the LC in migraine pathophysiology. Altered LC activity and functional connectivity were recorded through neuroimaging studies in migraineurs, with changes in the dorsal pontine activation and connectivity with the hypothalamus (Maniyar et al. 2014;Moulton et al. 2014). Migraine-involved pain molecules including CGRP and PACAP

were also located at the level of the LC [START_REF] Tajti | Neuropeptide Localization in the 'Migraine Generator' Region of the Human Brainstem[END_REF].

Moreover, the LC has been shown to respond to trigeminovascular activation, as shown by its increased activation following the administration of NTG in rats [START_REF] Tassorelli | Systemic Nitroglycerin Induces Fos Immunoreactivity in Brainstem and Forebrain Structures of the Rat[END_REF]. Also, it has been shown that the prophylactic effect of propranolol in a migraine model of inflammatory soup in rats was exerted through blockage of the chronic sensitization of descending controls of pain arising from the LC [START_REF] Boyer | Propranolol Treatment Prevents Chronic Central Sensitization Induced by Repeated Dural Stimulation[END_REF]). In addition, in a conscious rat model of trigeminal nociception, the LC and the dorsal raphe nucleus were both activated, both being participants in antinociceptive mechanisms (Ter Horst et al. 2001). A recent study assessed the impact of LC dysregulation in two validated preclinical models of migraine (Figure 12A) (Vila-Pueyo et al. 2019). They highlighted the antinociceptive role of LC inhibition in migraine-related pain. When LC signaling is decreased, there is an inhibition of dural-evoked trigeminal activation at the level of the TCC and hence nociceptive inhibition. Moreover, they showed a reduction in the threshold of CSD induction due to the decreased cortical LC noradrenergic signaling, impacting the occurrence of migraine aura (Figure 12B). Further showcasing of the role of the LC came about through the transcutaneous auricular vagus nerve stimulation in migraine patients, which lead to the modulatory decrease in the activity of the LC while increasing its connectivity with other regions (hippocampus, somatosensory cortex, and amygdala) (Y. [START_REF] Zhang | Transcutaneous Auricular Vagus Nerve Stimulation at 1 Hz Modulates Locus Coeruleus Activity and Resting State Functional Connectivity in Patients with Migraine: An FMRI Study[END_REF]. All that has been done so far to showcase the role of the LC in migraine, highlights the pivotal role of the LC and its noradrenergic projections in the pathophysiology of migraine.

IX. Neuroinflammation

Definition and its Role in Migraine Pathophysiology

Neuroinflammation is the reaction of the nervous system to noxious stimuli such as trauma, toxins, or injury that affect homeostasis leading to progressive neuronal death and carries a primary role in the pathophysiology of a plethora of neurological diseases [START_REF] Gilhus | Neuroinflammationa Common Thread in Neurological Disorders[END_REF]. The hallmark of neuroinflammation is attributed to the activation of adaptive immunity (the body learns to recognize the unique antigens of a pathogen, and thus formulates an antigen-specific response against it), microglia, and astrocytes [START_REF] Ransohoff | Microglial Physiology: Unique Stimuli, Specialized Responses[END_REF].

Over the last decades, the role of neuroinflammation in primary headache pathophysiology has been increasingly recognized. Indeed, data from pre-clinical models support the involvement of pro-inflammatory cytokines and immune cells, both in the induction and maintenance of migraine.

According to the current neurovascular theory described before, migraine attacks are related to hypothalamic activation, which is responsible for the stimulation of the Sp5C that, in turn, stimulates TG that releases CGRP. CGRP induces plasma protein extravasation together with dural mast cell activation, which releases histamine, 5-HT, NO, and pro-inflammatory cytokines, including IL-1, TNF-α, and IL-6. These inflammatory mediators contribute to the activation of dural trigeminal fibers, and enhance CGRP release, creating a positive feedback loop within the ganglion, therefore contributing to trigeminal pain transmission.

Besides mast cells, other cells are believed to contribute to meningeal neurovascular inflammation. For instance, activated macrophages can release proinflammatory cytokines. Also, dendritic cells along with T lymphocytes (for the most part memory CD4+ and CD3 T cells) in the dural vessels, and the subarachnoid space, have been reported as putative players in the pathophysiology of migraine [START_REF] Mcmenamin | Macrophages and Dendritic Cells in the Rat Meninges and Choroid Plexus: Three-Dimensional Localisation by Environmental Scanning Electron Microscopy and Confocal Microscopy[END_REF]. This is supported by evidence from experimental models of their role in mediating inflammatory pain [START_REF] Kashem | Nociceptive Sensory Fibers Drive Interleukin-23 Production from CD301b+ Dermal Dendritic Cells and Drive Protective Cutaneous Immunity[END_REF]. Resident glial cells also possess CGRP receptors, whose activation stimulates the production of TNF-α and IL-1, both of which amplify trigeminal nociception (Lars Edvinsson, Haanes, and Warfvinge 2019).

Based on the putative role of neuroinflammation in migraine coming from experimental animal models, several researchers investigated changes in cytokine levels and lymphocyte subsets in migraineurs, both in headache-free periods and during attacks. For instance, TNFα plays a role in the sensitization of trigeminal nerve fibers, the regulation of pain threshold, and the modulation of migraine perception [START_REF] Bruno | An Overview on Immune System and Migraine[END_REF]). TNFα has also shown an increase after the onset of migraine pain and a progressive decrease over time post-attack onset in migraineurs [START_REF] Perini | Plasma Cytokine Levels in Migraineurs and Controls[END_REF].

Changes of IL-6 and IL-10 in the blood of migraineurs were also shown with those of IL-6 being significantly higher during and between the attacks compared to healthy controls (Aydın et al. 2015). As for IL-10, they were the highest only during the attacks compared to the controls. Hence, migraine headache pathogenesis is affected by the contribution of cytokines including TNF-α, IL-6, IL-10, and IL-1β.

In migraine with aura, CSD may trigger neuroinflammation via the pannexin-1 channel opening and subsequent caspase-1 activation, which is responsible for the cleavage of IL-1β and IL-18. These cytokines, in turn, activate parenchymal neuroinflammatory signaling and nuclear factor-kappa B (NF-κB) activation in astrocytes [START_REF] Karatas | Spreading Depression Triggers Headache by Activating Neuronal Panx1 Channels[END_REF]). In addition, CSD rodent models using KCl cortical application, optogenetics, pinprick, and electrical stimulation show the induction of neuroinflammatory responses [START_REF] Takizawa | Temporal Profiles of High-Mobility Group Box 1 Expression Levels after Cortical Spreading Depression in Mice[END_REF][START_REF] Takizawa | Non-Invasively Triggered Spreading Depolarizations Induce a Rapid pro-Inflammatory Response in Cerebral Cortex[END_REF]. Although there is a debate surrounding the permeability of the BBB during migraine, CSD reportedly transiently activates the matrix metalloproteinase-9 temporarily breaking down the BBB, potentially showing that the vasoactive and intracellular nociceptive molecules induce inflammation and sneak their way through the BBB to reach the dura mater nociceptors (S.-P. Chen et al., 2017;[START_REF] Hougaard | Increased Brainstem Perfusion, but No Blood-Brain Barrier Disruption, during Attacks of Migraine with Aura[END_REF].

The Glial Cells

Glia is a non-neuronal, immune-like cell population that constitutes the vast majority of cells within the CNS. They comprise satellite glial cells in the ganglia, microglia, astrocytes, and oligodendrocytes within the spinal cord and brain. The anatomical co-localization of astrocytes and microglia with pre-and postsynaptic neurons forms a key site of interaction termed the "tetrapartite synapse" (De [START_REF] Leo | The Tetrapartite Synapse: Path to CNS Sensitization and Chronic Pain[END_REF]. Each cell within this functional unit reciprocally signals to another, contributing to a neuroimmune communication that allows glia to respond rapidly to disruptions in neuronal signaling. The reactivity state and control of astrocytes and microglia is therefore critical in maintaining healthy CNS activity.

Within this part, we will primarily focus on the role of astrocytes and microglia in migraine.

Astrocytes

The most abundant glial cells in the mammalian brain, astrocytes, play a central role in the maintenance of the function of the CNS (Liddelow and Barres 2017). They are broadly involved in water homeostasis, neuronal metabolic support, and BBB maintenance [START_REF] Parpura | Homeostatic Function of Astrocytes: Ca2+ and Na+ Signalling[END_REF][START_REF] Verkhratsky | The Special Case of Human Astrocytes[END_REF]. Furthermore, through astrocyte-microglia crosstalk, astrocytes modulate microglial phenotypes and phagocytosis and through astrocyte-neuron interactions, they regulate excitatory synaptic transmission [START_REF] Jha | Microglia-Astrocyte Crosstalk: An Intimate Molecular Conversation[END_REF]. In terms of structure, astrocytes are smaller in size compared to neurons, with a smaller soma, surrounded by various cytoplasmic extensions capable of connecting with up to 4 to 8 neurons for every single astrocyte [START_REF] Bushong | Protoplasmic Astrocytes in CA1 Stratum Radiatum Occupy Separate Anatomical Domains[END_REF][START_REF] Vasile | Human Astrocytes: Structure and Functions in the Healthy Brain[END_REF]. Identification of astrocytes is commonly done using the glial fibrillary acidic protein (GFAP) as a marker, which is an intermediate filament protein. Another marker used is the S100B (Ca 2+ binding protein) [START_REF] Brozzi | S100B Protein Regulates Astrocyte Shape and Migration via Interaction with Src Kinase *[END_REF].

In a process known as reactive astrogliosis, astrocytes undergo a series of phenotypic and functional alterations in response to painful stimulation and nerve injury. During this process, naïve astrocytes transform into reactive ones with specific phenotypic changes including (1) morphological changes (e.g. hypertrophy), (2) proliferation, (3) gene expression changes, (4) significant molecular changes, and (5) functional changes. Afterward, reactive astrocytes proliferate, migrate, and transform into scar-forming astrocytes [START_REF] Hara | Interaction of Reactive Astrocytes with Type I Collagen Induces Astrocytic Scar Formation through the Integrin-N-Cadherin Pathway after Spinal Cord Injury[END_REF]. High expressions of the astrocyte marker proteins (GFAP, β-catenin, nestin, and N-cadherin), are found in both reactive and scar-forming astrocytes however they do also have their personal marker genes (Figure 13). Adapted from (T. Li et al. 2019).

Reactive astrocytes can be further divided into toxic A1 astrocytes which induce rapid death of neurons and oligodendrocytes, and A2 astrocytes, which are neuroprotective and promote neuronal survival and tissue repair (Liddelow and Barres 2017). Both types can be identified according to their genetic expressions. In A1 astrocytes Complement 3 (C3), Complement factor B (CFB), and myxovirus resistance-1 (MX1) are the most characteristic and predominately upregulated genes that are not expressed in A2, while the S100 protein family member S100A10 has been identified as a specific marker of A2 astrocytes (Liddelow et al. 2017). Reactive astrogliosis can increase neuroprotection and nutritional support for damaged neurons. Furthermore, activated astrocytes can reconstruct the damaged BBB and limit the infiltration of peripheral leukocytes. Thus, astrogliosis is an initial defense mechanism for repairing damage.

The transformation of astrocytes from their normal state to reactive phenotypes encompasses multiple intercellular and intracellular signaling processes, which can release various molecules by multiple cell types, including neurons and other glial cells.

Released signaling molecules include gene transcription factors [extracellular signalregulated kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3)], pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), and proteins (GFAP and connexins) (T. Li et al. 2019).

Microglia

Microglia are cells with macrophage-like abilities residing in 5-10% of the parenchyma of the brain and spinal cord. They continuously survey the environment, clearing debris and dead cells caused by potential infection or damage (Gomez [START_REF] Perdiguero | Tissue-Resident Macrophages Originate from Yolk-Sac-Derived Erythro-Myeloid Progenitors[END_REF], and form interactions with neurons, astrocytes, and oligodendrocytes to modulate synaptic neurotransmission [START_REF] Bachiller | Microglia in Neurological Diseases: A Road Map to Brain-Disease Dependent-Inflammatory Response[END_REF][START_REF] Matejuk | Crosstalk Between Astrocytes and Microglia: An Overview[END_REF]. When activated under neuroinflammatory conditions, microglia migrate toward the site of damage exerting beneficial or detrimental effects through the release of inflammatory mediators, neurotrophic factors, and reactive oxygen species [START_REF] Wolf | Microglia in Physiology and Disease[END_REF].

Their Dual Role and Contribution in Persistent Pain

Following injury or nociceptive events, compounds released from injured tissue and neuronal neurotransmitters stimulate microglia and astrocytes, which increase their expression and secretion of various pro-inflammatory cytokines and chemokines. This pro-inflammatory response is important in protecting against challenges that disrupt the homeostatic balance of the CNS. However, under certain conditions, glial reactivity is not advantageous and can instead be detrimental to neuronal function, such as during the manifestation of persistent pain.

Astrogliosis is a double-edged sword. Although the present evidence suggests that there exists an important neuroprotective and reparative role for both reactive astrocytes and microglia in the initial stage of nerve injuries, however, their inhibitory effects on functional recovery after injury are undeniable. In particular, they may encourage the development and maintenance of chronic pain. Their activation has been observed in neuropathic pain models and spinal cord infection models [START_REF] Cao | Spinal Glial Activation Contributes to Pathological Pain States[END_REF][START_REF] Raghavendra | Inhibition of Microglial Activation Attenuates the Development but Not Existing Hypersensitivity in a Rat Model of Neuropathy[END_REF]. Moreover, intrathecal transfer of highly reactive microglia alone, or injection of their pro-inflammatory products (such as IL-1β

and TNFα) into naive animals, can induce symptoms of neuropathic pain [START_REF] Tsuda | P2X4 Receptors Induced in Spinal Microglia Gate Tactile Allodynia after Nerve Injury[END_REF]. IL-1β is upregulated in spinal microglia and astrocytes following peripheral nerve injury, accumulating evidence of its involvement in pain sensitization [START_REF] Kiguchi | Chemokines and Cytokines in Neuroinflammation Leading to Neuropathic Pain[END_REF]. TNF-α plays an important role in the initiation and development of persistent pain and central sensitization and is primarily produced by microglia [START_REF] Ji | Glia and Pain: Is Chronic Pain a Gliopathy?[END_REF]. Both mechanical and thermal hypersensitivities were induced after an intrathecal injection of exogenous TNF, while prevention of the development of neuropathic pain is done after an intrathecal injection of anti-TNF antibodies prior to a peripheral nerve injury [START_REF] Clark | Neuropathic Pain and Cytokines: Current Perspectives[END_REF].

During the development of pain, the responses of microglia are typically early and transient, while the activation of astrocytes is later and remains longer than microglia. At the level of the dorsal horn, glial-derived pro-inflammatory mediators enhance nociceptive signaling by facilitating glutamatergic neurotransmission (Figure 14). IL-1β has been shown to increase presynaptic release of glutamate and together with TNFα and IFNγ, increases postsynaptic N-methyl-D-aspartic (NMDA) and AMPA receptor currents. The aberrant uptake and/or release of glutamate, as well as the enhanced activity of its postsynaptic receptors, can contribute to excessive nociceptive signaling reaching the brain [START_REF] Dodds | Glial Contributions to Visceral Pain: Implications for Disease Etiology and the Female Predominance of Persistent Pain[END_REF]):

To prevent the persistence of pathological pain, minocycline can be used as a microglia inhibitor, although it does not reverse the hyperalgesia that is established after nerve injury/inflammation [START_REF] Raghavendra | Inhibition of Microglial Activation Attenuates the Development but Not Existing Hypersensitivity in a Rat Model of Neuropathy[END_REF] However, intrathecal injections of astrocyte inhibitors (such as valerine, fluorocitrate, and l-1amino-hexanedioic acid), could reverse mechanical cutaneous hypersensitivity and reduce the maintenance of abnormal pain [START_REF] Cao | Spinal Glial Activation Contributes to Pathological Pain States[END_REF]. These studies indicate the role of microglia as a contributor to the initiation of mechanical allodynia, while astrocytes could be responsible for its maintenance.

.

Their Role in Migraine Pathophysiology

Unfortunately, few studies have focused on the role of astrocytes and microglia in migraine pathogenesis. FHM type 2 is caused by the Atp1a2 gene, encoding the 2 subunits of the Na+/K+-dependent adenosine triphosphatase [START_REF] Fusco | Haploinsufficiency of ATP1A2 Encoding the Na+/K+ Pump Alpha2 Subunit Associated with Familial Hemiplegic Migraine Type 2[END_REF], that is exclusively expressed in astrocytes [START_REF] Melone | Heterogeneity of Astrocytic and Neuronal GLT-1 at Cortical Excitatory Synapses, as Revealed by Its Colocalization With Na+/K+-ATPase α Isoforms[END_REF]. This data provide evidence that astrocytes may have a role in the diffuse alterations in brain function that occur during a migraine attack. Moreover, in a mouse model of migraine, astrocyte dysfunction in the cingulate gyrus leads to prominent hypersensitivity and could potentially be involved in sensitization in familial migraine [START_REF] Romanos | Astrocyte Dysfunction Increases Cortical Dendritic Excitability and Promotes Cranial Pain in Familial Migraine[END_REF].

Interestingly, astrocytes have shown waves of activity with similar patterns to those of CSD in terms of temporal and spatial characteristics, postulating that these waves could be initiating a propagation of changes in the vascular changes and brain activity observed in migraineurs (A. [START_REF] Charles | Cortical Spreading Depression-New Insights and Persistent Questions[END_REF]. In addition, the dysfunction of glutamate clearance by cortical astrocytes also leads to the potential facilitation of CSD initiation [START_REF] Capuani | Defective Glutamate and K+ Clearance by Cortical Astrocytes in Familial Hemiplegic Migraine Type 2[END_REF]. From here, growing evidence is emerging to further elucidate the contribution of astrocytes in migraine pathogenesis, and further work has to be put into the potential comprehension of their role.

Regarding microglia, recurrent administrations of NTG induce central sensitization by activation of microglial cells [START_REF] Long | Microglia P2X4R-BDNF Signalling Contributes to Central Sensitization in a Recurrent Nitroglycerin-Induced Chronic Migraine Model[END_REF]. Precisely through the microglial P2X4R/BDNF (Brain-derived neurotrophic factor) pathway whereby the blockade of P2X4Rs, which are a part of the purinergic P2 receptors that have been widely studied in neuropathic pain, prevented both hyperalgesia and the release of CGRP [START_REF] Tsuda | Microglia and Intractable Chronic Pain[END_REF]. CSD is one of the stimuli for microglial activation, as it is a dysregulated slow propagating wave of neuronal and glial activation that leads to the release of a plethora of microglia-activating molecules [START_REF] Borgdorff | Arguments against the Role of Cortical Spreading Depression in Migraine[END_REF][START_REF] Shibata | Exploring the Role of Microglia in Cortical Spreading Depression in Neurological Disease[END_REF]. Post-activation microglia take on two different phenotypes ( M1 and M2) depending on their activating agent (S. [START_REF] Zhao | Regulation of Microglial Activation in Stroke[END_REF]. Linked with the production of proinflammatory cytokines, the M1 phenotype, releases IL-1, TNF-α, NO, and ROS. On the other hand, the M2 phenotype is the one responsible for the repair and regeneration of tissue through the release of the anti-inflammatory cytokines, IL-13, IL-4, and IL-10 [START_REF] Kraft | Features of Microglia and Neuroinflammation Relevant to Environmental Exposure and Neurotoxicity[END_REF]. The activation of the microglia for prolonged durations has shown adverse effects in other neurodegenerative and neoplastic diseases, and potentially in migraine as well [START_REF] Taipa | Inflammatory Pathology Markers (Activated Microglia and Reactive Astrocytes) in Early and Late Onset Alzheimer Disease: A Post Mortem Study[END_REF]. So far, we have concrete evidence of the contribution of microglia in migraine precisely chronic migraine and its central sensitization, however further work is to be undertaken to further understand its mechanistic contribution

(

X. Aryl Hydrocarbon Receptors

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor of the basic helix-loop-helix (bHLH)-PER/ARNT(AhR Nuclear Translocator)/SIM homology family. They are involved in many signaling pathways such as the response to environmental pollutants (xenobiotics), hypoxia, the circadian rhythm, and neural development. Moreover, AhR mediates the regulation of immunity, maintains gut homeostasis, and ameliorates inflammation.

The original discovery of AhR was in hepatocytes in the 1970s as the mediator of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD/dioxin) toxicity [START_REF] Poland | Stereospecific, High Affinity Binding of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin by Hepatic Cytosol. Evidence That the Binding Species Is Receptor for Induction of Aryl Hydrocarbon Hydroxylase[END_REF]. Overt dioxin exposure in humans leads to a chronic inflammatory condition characterized by persistent painful skin lesions. AhR structure is highly conserved between species and expressed in vertebrates with different isoforms. In mammals, a unique AhR isoform, AhR1, has been identified [START_REF] Hahn | Unexpected Diversity of Aryl Hydrocarbon Receptors in Non-Mammalian Vertebrates: Insights from Comparative Genomics[END_REF].

Protein Structure

AhR Expression

Many studies have shown that AhR is expressed ubiquitously, however, variations exist depending on the tissue. Its expression begins during embryonic development, primarily in the neuroepithelium and the heart; then in the liver, and finally in other tissues including kidneys, lungs, muscles, and epidermis. In adult humans, the highest levels of expression are found in the lung and liver [START_REF] Dolwick | Cloning and Expression of a Human Ah Receptor CDNA[END_REF]).

In the brain, AhR messenger RNA (mRNA) (together with ARNT mRNA)

shows specific temporal and spatial patterns of expression in the mouse cortex, hippocampus, cerebellum, and olfactory bulb (E. [START_REF] Kimura | Embryonic and Postnatal Expression of Aryl Hydrocarbon Receptor MRNA in Mouse Brain[END_REF]. The brainstem including the LC (E. Kimura et al. 2021), the thalamus (including Po), and several nuclei of the hypothalamus have markedly higher AhR amounts than other regions of the brain. These regions include the ventromedial hypothalamus, paraventricular nucleus, suprachiasmatic nucleus, and dorsal and median raphe nuclei (Petersen et al. 2000). Interestingly, AhR mRNA levels were low in the spinal trigeminal tract, amygdala, septal region, and forebrain; and undetectable in the basomedial nucleus, superior olivary complex, vestibular nuclei, NRM, and lateral preoptic area (Petersen et al. 2000).

AhR protein is present in neurons, astrocytes, oligodendrocytes, and microglia [START_REF] Barroso | The Aryl Hydrocarbon Receptor and the Gut-Brain Axis[END_REF], and is involved in the regulation of neurogenesis, neuronal migration, and neurite elongation during development in mice and humans [START_REF] Dever | Aryl Hydrocarbon Receptor (AhR) Deletion in Cerebellar Granule Neuron Precursors Impairs Neurogenesis[END_REF]E. Kimura and Tohyama 2017). The regulation of its expression depends not only on internal stimuli, but also on traumatic injury, exposure to xenobiotics, and endocrine disruptors such as bisphenol A.

The AhR Signaling Pathways

AhR activation results in its translocation to the cell nucleus and control of the expression of target genes harboring AhR-responsive DNA elements [known as xenobiotic response elements (XRE)] in their regulatory regions. Additional nongenomic AhR signaling pathways have also been described.

XRE-dependent Control of Gene Expression by AhR

When inactive, AhR is complexed with the cellular-sarcoma (c-Src) kinase, cochaperone p23, two heat shock protein 90 (Hsp90) chaperons, and X-associated protein 2 (XAP2) also known as the AhR-interacting protein (AIP)/ARA9) at the level of the cytoplasm. This chaperone complex keeps AhR inactive, prevents proteasomal degradation, and keeps it in a high-affinity state for its ligands. Upon agonist binding, AhR and some components of the chaperone complex translocate to the nucleus, whereby further dissociation of the other parties (p23, XAP2, and the second Hsp90)

allows for AhR to dimerize with ARNT (Figure 16). The AhR/ARNT heterodimer then binds to XREs and hence regulates the transcription of target genes. AhR is a regulator of a plethora of prototypic genes such as Cytochrome P450 Family 1 Subfamily A Member 1 (CYP1A1), Cytochrome P450 Family 1 Subfamily B Member 1 (CYP1B1), and the AhR repressor (AhRR). AhRR competes with the AhR-ligand complex for its interaction with ARNT, limiting the availability of ARNT for AHR-ligand binding and consequently providing an additional negative regulatory mechanism of AhR-driven gene expression [START_REF] Sakurai | The Crystal Structure of the AhRR-ARNT Heterodimer Reveals the Structural Basis of the Repression of AhR-Mediated Transcription[END_REF].

XRE-independent Control of Gene Expression by AhR

AhR can also interact with additional transcription factors to target their specific binding sites (Rothhammer and Quintana 2019). For example, AhR controls nuclear NF-κB and its dependent transcriptional programs indirectly through the suppression of cytokine signaling 2 -dependent mechanisms and directly by interacting with RELA, RELB, and other members of the NF-κB signaling complex [START_REF] Salisbury | The AhR and NF-ΚB/Rel Proteins Mediate the Inhibitory Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on the 3' Immunoglobulin Heavy Chain Regulatory Region[END_REF][START_REF] Vogel | Cross-Talk between Aryl Hydrocarbon Receptor and the Inflammatory Response: A Role for Nuclear Factor-ΚB[END_REF]). Similarly, AhR has been reported to regulate the activation of STAT proteins that play central roles in the regulation of immune responses (A. [START_REF] Kimura | Aryl Hydrocarbon Receptor Regulates Stat1 Activation and Participates in the Development of Th17 Cells[END_REF][START_REF] Quintana | An Endogenous Aryl Hydrocarbon Receptor Ligand Acts on Dendritic Cells and T Cells to Suppress Experimental Autoimmune Encephalomyelitis[END_REF]. This interaction leads to the modulation of several outcomes including inflammation, proliferation, differentiation, and endocrine disruption [START_REF] Kim | The RelA NF-ΚB Subunit and the Aryl Hydrocarbon Receptor (AhR) Cooperate to Transactivate the c-Myc Promoter in Mammary Cells[END_REF][START_REF] Ohtake | Modulation of Oestrogen Receptor Signalling by Association with the Activated Dioxin Receptor[END_REF][START_REF] Procházková | The Interplay of the Aryl Hydrocarbon Receptor and β-Catenin Alters Both AhR-Dependent Transcription and Wnt/β-Catenin Signaling in Liver Progenitors[END_REF].

Additional AhR Signaling Mechanisms

In addition, when a ligand binds to AhR, this modulates AhR's ability to form interactions with different proteins, such as kinases, Kruppel-like factor 6 (KLF6), retinoblastoma protein (RB), and Cullin 4B (CUL4B) (D. P. [START_REF] Jackson | Ah Receptor-Mediated Suppression of Liver Regeneration through NC-XRE-Driven P21Cip1 Expression[END_REF][START_REF] Tomkiewicz | The Aryl Hydrocarbon Receptor Regulates Focal Adhesion Sites through a Non-Genomic FAK/Src Pathway[END_REF]). The interactions with those molecules activate nongenomic pathways. For instance, exposure to TCDD in rats, leads to a rapid increase in the intracellular concentration of Ca 2+ , activating PKC [START_REF] Hanneman | Stimulation of Calcium Uptake in Cultured Rat Hippocampal Neurons by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin[END_REF]. PKC then phosphorylates phospholipase A2 (cPLA2) leading to the production of arachidonic acid. Following the dissociation of the cytoplasmic AhR complex, c-Src is released, activating the MAPKs, and leads to the transcription of the COX2 mRNA (Figure 16). The latter's protein uses arachidonic acid to produce prostaglandins and hence causes inflammation [START_REF] Matsumura | The Significance of the Nongenomic Pathway in Mediating Inflammatory Signaling of the Dioxin-Activated Ah Receptor to Cause Toxic Effects[END_REF]. Moreover, the activated c-Src induces the activation of focal adhesion kinase (FAK) leading to the modification of cellular adhesion [START_REF] Tomkiewicz | The Aryl Hydrocarbon Receptor Regulates Focal Adhesion Sites through a Non-Genomic FAK/Src Pathway[END_REF].

AhR Ligands

A multitude of structurally diverse compounds constitute the ligands of AhR, including endogenous substances such as intermediary and microbial metabolites of tryptophan (TRP), and exogenous compounds such as drugs, alkaloids, and synthetic compounds. Differences in the binding affinity exist between these ligands, in addition to differences in chemical structures. In terms of their intrinsic activity, AhR ligands are either agonists or antagonists.

AhR Agonists

AhR agonists are multiple and can be classified into exogenous and endogenous agonists. The main source of exposure in animals and humans to exogenous ligands comes from their diet (90%). They can be grouped according to their origin: "synthetic"

or natural [START_REF] Denison | Activation of the Aryl Hydrocarbon Receptor by Structurally Diverse Exogenous and Endogenous Chemicals[END_REF]. Synthetic exogenous agonists include To date, the endogenous (physiological) AhR agonists are not yet known with certainty. However, many candidates have been identified and are regulators of the expression of the AhR-dependent gene cluster. They include TRP derivatives and indoles, arachidonic acid, certain leukotrienes (lipoxin A4), and heme metabolites (bilirubin and biliverdin) (Table 2). Among these ligands, TRP and its derivatives have been the most studied. TRP is one of the essential amino acids, naturally synthesized by microorganisms and plants, and cannot be synthesized by the human body. Its derivatives are formed in three main ways: kynurenine, serotonin, and indole pathways (a specific section will be dedicated to TRP metabolism below). Reports have shown that kynurenine is a highly potent agonist of AhR [START_REF] Seok | Trace Derivatives of Kynurenine Potently Activate the Aryl Hydrocarbon Receptor (AHR)[END_REF]. For instance, when binding to AhR, kynurenine, considered a high-affinity ligand, stimulates the receptor which enhances immunosuppression [START_REF] Salminen | Role of Indoleamine 2,3-Dioxygenase 1 (IDO1) and Kynurenine Pathway in the Regulation of the Aging Process[END_REF].

Indole produced by gut bacteria has demonstrated AhR agonist activity in yeast assay [START_REF] Bansal | The Bacterial Signal Indole Increases Epithelial-Cell Tight-Junction Resistance and Attenuates Indicators of Inflammation[END_REF], although, in certain specific conditions, it also behaves as an AhR antagonist (Hubbard et al. 2015;U.-H. Jin et al. 2014). Other compounds derived from TRP such as indole-3-acetic acid, tryptamine, and 3-indoxyl sulfate (I3S) have also shown an agonistic role [START_REF] Zelante | Tryptophan Catabolites from Microbiota Engage Aryl Hydrocarbon Receptor and Balance Mucosal Reactivity via Interleukin-22[END_REF]. Furthermore, 2-(1′H-indole-3′carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is considered an endogenous AhR ligand (J. [START_REF] Song | A Ligand for the Aryl Hydrocarbon Receptor Isolated from Lung[END_REF].

AhR Antagonists

AhR is bound by structurally different phytochemicals, one of which being brevifolincarboxylic acid which inhibits their activity (Table 2) [START_REF] Amakura | Screening of the Inhibitory Effect of Vegetable Constituents on the Aryl Hydrocarbon Receptor-Mediated Activity Induced by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin[END_REF].

Moreover, green tea is a potential inhibitor of the expression of AhR-regulated genes [START_REF] Fukuda | Catechins in Tea Suppress the Activity of Cytochrome P450 1A1 through the Aryl Hydrocarbon Receptor Activation Pathway in Rat Livers[END_REF]Han et al. 2012). Through further comprehension of the role of AhR and their respective pathways, the interest in constructing novel AhR antagonists arose through structural modifications intended to improve both pharmacokinetics and pharmacological properties. For instance, a potent oral AhR antagonist, BAY-2416964 was able to inhibit the receptor's activity in both humans and mice by preventing AhRmediated signaling (L. Sun 2021). Another antagonist, 6,2',4'-trimethoxyflavone (TMF) was found to be a competing antagonist with other agonists of AhR possessing the characteristics of allowing further dissection of the role of AhR [START_REF] Murray | Antagonism of Aryl Hydrocarbon Receptor Signaling by 6,2',4'-Trimethoxyflavone[END_REF].

The Physiological Significance of AhR

Several AhR has shown its involvement in a plethora of inflammatory disorders with its role being either of a pro or anti-inflammatory nature depending on the tissue and cell type (Juricek and Coumoul 2018). In rheumatoid arthritis, activation of AhR contributes to disease progression and severity [START_REF] Fu | Shared Epitope-Aryl Hydrocarbon Receptor Crosstalk Underlies the Mechanism of Gene-Environment Interaction in Autoimmune Arthritis[END_REF]. Furthermore, in AhR knockout mice, low levels of pro-inflammatory cytokines were detected in the serum, concomitant with a decrease in disease severity [START_REF] Nakahama | Aryl Hydrocarbon Receptor Deficiency in T Cells Suppresses the Development of Collagen-Induced Arthritis[END_REF].

Cases of IBD, such as Crohn's Disease, have shown low expression levels of AhR [START_REF] Qiu | Aryl Hydrocarbon Receptor Promotes RORγt + Group 3 ILCs and Controls Intestinal Immunity and Inflammation[END_REF]. Moreover, in IBD patients, decreased levels of AhR ligands compared to their controls have also been reported (Rothhammer et al. 2016). The administration of AhR ligands such as TCDD can ameliorate the symptoms of certain IBD with a decrease in the production of pro-inflammatory cytokines and increasing anti-inflammatory cytokine productions (such as IL-22) (Q. Wang et al., 2018).

As previously mentioned, AhR is expressed at the level of different CNS cellular populations. However, their roles differ according to at which cells they are expressed.

Activation of astrocytic AhR by endogenous indoles reduces inflammation and has a neuroprotective action (Rothhammer et al. 2016). However, the activation of neuronal AhR plays a rather pro-nociceptive role in facilitating an inflammatory state (Ntranos et al. 2022). The role of AhR in pain has been mildly studied, whereby in one animal model of chronic constrictive nerve injury there was an augmentation of AhR expression, and the deletion of AhR worsened the damage, while the administration of an AhR agonist counteracted such changes (Sheu et al. 2022). So far concerning migraine, AhR's influence on the pathophysiology of migraine has never been addressed and therefore remains to be identified.

XI-The Metabolism of Tryptophan

Tryptophan, an Essential Amino Acid

TRP is an essential amino acid, that can only be obtained through the daily diet ([tuna, cheese, milk, turkey, oats, seeds, and nuts [START_REF] Shabbir | Effect of Diet on Serotonergic Neurotransmission in Depression[END_REF]], necessary for various metabolic reactions, and for the production and maintenance of proteins, enzymes, muscles, and neurotransmitters. It is an aromatic amino acid from the erythrose 4-phosphate and phosphoenolpyruvate family [START_REF] Palego | Tryptophan Biochemistry: Structural, Nutritional, Metabolic, and Medical Aspects in Humans[END_REF]. Levels of circulating plasma TRP are heavily influenced by geographic and cultural diet practices, physical activity, and stress (K. Gao et al. 2020;[START_REF] Yu | Increases in Plasma Tryptophan Are Inversely Associated with Incident Cardiovascular Disease in the Prevención Con Dieta Mediterránea (PREDIMED) Study[END_REF]. The majority of ingested TRP is absorbed by the small intestine, and its availability is affected by the simultaneous absorption of other neutral amino acids ( valine, leucine, isoleucine, serine, histidine, glutamine, asparagines, phenylalanine, tyrosine) [START_REF] Agus | Gut Microbiota Regulation of Tryptophan Metabolism in Health and Disease[END_REF]. Dietary carbohydrates induce an influence on TRP uptake since there is a preferable capture of non-TRP neutral amino acids due to the induction of insulin release [START_REF] Keszthelyi | Understanding the Role of Tryptophan and Serotonin Metabolism in Gastrointestinal Function[END_REF]. When insulin is elevated, this promotes the uptake of long neutral amino acids except for TRP to the skeletal muscles, hence increasing the plasma TRP to long neutral amino acid ratio and hence the influx of TRP to the brain. In peripheral blood circulation, TRP is either albumin-bound (80-90%) or freely solubilized. The differential uptake of amino acids by the skeletal muscles is due to the fact that TRP is bound to albumin in the plasma. Free TRP proceeds to the CNS via the L-type amino acid transporter (Lat1) which is the chaperon responsible for the transportation of large neutral amino acids across the BBB [START_REF] Pardridge | Transport of Tryptophan into Brain from the Circulating, Albumin-Bound Pool in Rats and in Rabbits[END_REF].

Typically, individuals consume between 900 to 1000 mg of TRP daily even though the recommended daily allowance of TRP is between 250 to 425 mg for adults which corresponds to 3.5 to 6.0 mg/kg of body weight per day [START_REF] Richard | L-Tryptophan: Basic Metabolic Functions, Behavioral Research and Therapeutic Indications[END_REF]. A TRP deficiency is not quite common naturally but can be attributed to insufficient dietary protein consumption (veganism, vitamin B6, high sugar intake, or excessive consumption of alcohol). There are other cases involving the excretion of TRP in the urine. For example, the Hartnup disease, a recessive genetic disorder in which the renal and intestinal transport of neutral amino acids is defective, patients are likely to have TRP deficiency due to increased urinary excretion [START_REF] Ganapathy | Protein Digestion and Absorption[END_REF]. Moreover, in certain cases, TRP might not be fully catabolized by the intestines.

Patients with histories of inflammatory gut disorders such as IBD, have impairments in their intestinal permeability [START_REF] Chang | Impaired Intestinal Permeability Contributes to Ongoing Bowel Symptoms in Patients With Inflammatory Bowel Disease and Mucosal Healing[END_REF]. This malfunctioning creates a cascade of release of pro-inflammatory mediators that modifies the microbiota profile (Al [START_REF] Bander | The Gut Microbiota and Inflammation: An Overview[END_REF]). Hence, potentially modifying the catabolism of TRP by the gut.

Metabolic Pathways of Tryptophan

TRP is metabolized by three major metabolic pathways: 95% is metabolized along the kynurenine pathway; approximately 1-2 % are used for serotonin synthesis, and the rest are metabolized by the gut microbiota to obtain indole and its respective derivatives [START_REF] Agus | Gut Microbiota Regulation of Tryptophan Metabolism in Health and Disease[END_REF][START_REF] Muneer | Kynurenine Pathway of Tryptophan Metabolism in Neuropsychiatric Disorders: Pathophysiologic and Therapeutic Considerations[END_REF][START_REF] Richard | L-Tryptophan: Basic Metabolic Functions, Behavioral Research and Therapeutic Indications[END_REF].

Kynurenine Pathway

Approximately 95% of TRP is metabolized by 2,3-dioxygenase (TDO), giving rise to kynurenine while the remainder of TRP is metabolized in the brain, liver, and gastrointestinal tract by indoleamine 2,3-dioxygenase (IDO) (Figure 18). Kynurenine is then catabolized giving rise to two neuroactive inflammatory molecules: quinolinic acid and kynurenic acid (KYNA). Kynurenine catabolites acting centrally are normally produced locally and rarely cross the BBB. KYNA is an NMDA receptor antagonist at the glycine binding site acting as a neuroprotective factor, while quinolinic acid acts as an NMDA agonist and is considered neurotoxic [START_REF] Guillemin | Expression of Indoleamine 2,3-Dioxygenase and Production of Quinolinic Acid by Human Microglia, Astrocytes, and Neurons[END_REF]. Further catabolizing of quinolinic acid gives NAD+ and niacin which are two active molecules in other primary cellular metabolic processes. The kynurenine pathway and its derived metabolites exert their function at the level of the nervous, endocrine, and immune systems, with anti-inflammatory, anti-oxidative, and/or neuroprotective/neurotoxic properties [START_REF] Tsuji | The Tryptophan and Kynurenine Pathway Involved in the Development of Immune-Related Diseases[END_REF].

Serotonin Pathway

The 5-HT synthesis from TRP occurs predominantly at the level of the gastrointestinal tract (90%) and at a minor level in the CNS (10%) (Boadle-Biber 1993).

The initial rate-limiting step is catalyzed by tryptophan hydroxylase (Figure 19), which presents two isoforms, isoform 1 expressed by the enterochromaffin cells within the gut, and isoform 2 expressed by the serotoninergic neurons of the CNS located in the raphe nuclei mainly. These enzymes convert TRP to 5-hydroxytryptophan (5-HTP) which is then converted to serotonin by the L-amino acid decarboxylase. Serotonin can be further converted to melatonin, a primary regulator of circadian rhythms and sleep initiation within the pineal gland [START_REF] Cassone | Effects of Melatonin on Vertebrate Circadian Systems[END_REF]

Indole Pathway

To obtain indole, several bacterial clusters use TRP through the action of the enzyme tryptophanase. The cluster consists of bacteria involved in the development of various neuropsychiatric disorders. Some of the bacteria involved in TRP metabolism include the phyla Actinobacteria, Firmicutes, Bacteroidetes, Proteobacteria, and Fusobacteria [START_REF] Kaur | Tryptophan Metabolism by Gut Microbiome and Gut-Brain-Axis: An in Silico Analysis[END_REF][START_REF] Roager | Microbial Tryptophan Catabolites in Health and Disease[END_REF]. TRP is converted to indole-3-propionic acid (IPA) through the cascade involving the conversion of TRP to indole-3-pyruvic acid (IPYA) which is then converted to indole-3-lactic acid (ILA)

and anholocyclic acid (IA) before being converted to IPA (Figure 20). In addition, using other TRP degrading enzymes, different metabolic end products are created by microbes including indole-3-acetaldehyde (IAAld) which is formed from tryptamine, and indole-3-aldehyde (IAld) which is derived from IAAld. Furthermore, TRP can be directly converted to indole. In terms of their function, indoles regulate gut insulin secretion, regulate inflammatory processes, drug resistance, and toxicity, and maintain the health and youth of animals [START_REF] Chimerel | Bacterial Metabolite Indole Modulates Incretin Secretion from Intestinal Enteroendocrine L Cells[END_REF][START_REF] Powell | Indoles from the Commensal Microbiota Act via the AHR and IL-10 to Tune the Cellular Composition of the Colonic Epithelium during Aging[END_REF].

The Metabolism of TRP and its Role in the Inflammation

The metabolism of TRP is linked to aging and generates metabolites that modulate inflammation, maintain energy homeostasis, and influence behavior [START_REF] Cervenka | Kynurenines: Tryptophan's Metabolites in Exercise, Inflammation, and Mental Health[END_REF]. TRP metabolites also have a role in the regulation of tissue damage and inflammation. Serotonin is one example, which has been linked to intestinal inflammation (Spohn and Mawe 2017); KYNA, which induces antiinflammatory changes in adipose tissue [START_REF] Agudelo | Kynurenic Acid and Gpr35 Regulate Adipose Tissue Energy Homeostasis and Inflammation[END_REF]); 3-hydroxy anthranilic acid and cinnabarinic acid, (two other Kynurenine metabolites), which have been linked

to vascular inflammation (P. Song et al. 2017) and autoimmune encephalomyelitis [START_REF] Fazio | Cinnabarinic Acid, an Endogenous Agonist of Type-4 Metabotropic Glutamate Receptor, Suppresses Experimental Autoimmune Encephalomyelitis in Mice[END_REF]; NAD + , which protects from renal kidney damage (Poyan Mehr et al.

2018), and indoles, which are a pivotal player in neuronal and gastro-intestinal inflammation [START_REF] Roager | Microbial Tryptophan Catabolites in Health and Disease[END_REF]. Modulations in the levels of TRP, such as its deficiency has been shown to compromise immune responses and impair disease resistance in teleost fish [START_REF] Machado | Dietary Tryptophan Deficiency and Its Supplementation Compromises Inflammatory Mechanisms and Disease Resistance in a Teleost Fish[END_REF]. Furthermore, gut health and intestinal immunity require sufficient dietary TRP for efficient immunological response and intestinal homeostasis (J. [START_REF] Gao | Impact of the Gut Microbiota on Intestinal Immunity Mediated by Tryptophan Metabolism[END_REF]. TRP metabolites are known to support gut microbiota, microbial metabolism, the host's immune system, and host-microbiota synergy [START_REF] Lamas | CARD9 Impacts Colitis by Altering Gut Microbiota Metabolism of Tryptophan into Aryl Hydrocarbon Receptor Ligands[END_REF]. Indeed, intestinal inflammation and colitis could occur due to disruptions in the gut lactobacillus strains responsible for TRP metabolism [START_REF] Lamas | CARD9 Impacts Colitis by Altering Gut Microbiota Metabolism of Tryptophan into Aryl Hydrocarbon Receptor Ligands[END_REF].

The TRP Metabolism in the Migraine Pathophysiology

Disturbances in the levels of TRP can lead to imbalances in the synthesis of 5-HT and melatonin in the brain, potentially playing a role in the pathophysiology of a plethora of neurodegenerative and neuropsychiatric disorders [START_REF] Comai | Chapter Five -Tryptophan in Health and Disease[END_REF].

Animal models of TRP depletion (acute and chronic) have demonstrated the pivotal role of TRP in different pathologies.

Acute TRP depletion (ATD), uses a combination of a low TRP diet and a TRPdeficient protein load containing large amounts of the other large neutral amino acids to produce maximal CNS TRP depletion. It is attained using a mixture 7-amino-acid ATD mixture (L-phenylalanine, L-leucine, L-isoleucine, L-methionine, L-valine, L-threonine, and L-lysine) as described by Moja et al., which is associated with a marked reduction in plasma TRP levels [START_REF] Moja | Decrease in Plasma Tryptophan after Tryptophan-Free Amino Acid Mixtures in Man[END_REF]). This protocol was later modified for administration to young people (termed Moja-De) with a body-weight-adapted dosing scheme, and a lower amount of methionine relative to conventional mixtures (to reduce unwanted side effects). TRP competes with the other large neutral amino acids for uptake by the Lat1 from the plasma to the brain [START_REF] Fernstrom | The Ingestion of Different Dietary Proteins by Humans Induces Large Changes in the Plasma Tryptophan Ratio, a Predictor of Brain Tryptophan Uptake and Serotonin Synthesis[END_REF]). The ATD model has been well established in mice and humans showing a robust decrease in the peripheral and central levels of both TRP and serotonin [START_REF] Biskup | Effects of Acute Tryptophan Depletion on Brain Serotonin Function and Concentrations of Dopamine and Norepinephrine in C57BL/6J and BALB/CJ Mice[END_REF][START_REF] Wong | Effects of Dietary Acute Tryptophan Depletion (ATD) on NPY Serum Levels in Healthy Adult Humans Whilst Controlling for Methionine Supply-A Pilot Study[END_REF].

Chronic TRP depletion (CTD) has previously and reproducibly induced neurochemical and behavioral alterations in rats (Cahir et al. 2007) and mice (Browne et al. 2012). It relies on the same concept as the ATD amino acid drink, but rather administering dietary regimens (food) lacking in TRP for a longer period of time. CTD has shown stronger effects, reducing peripheral and central levels of both TRP and serotonin approximately 30 days post-diet (Browne et al. 2012). Moreover, long-term TRP-depleting diets lead to changes in serotonergic receptors in animals, for instance, increasing serotonin 5-HT2A receptor density (Cahir et al. 2007).

When it comes to migraine, changes in the levels of TRP are documented with a prominent decrease in the serum and plasma levels of TRP migraineurs compared to healthy controls [START_REF] D'andrea | Tryptamine Levels Are Low in Plasma of Chronic Migraine and Chronic Tension-Type Headache[END_REF][START_REF] Ren | Low Levels of Serum Serotonin and Amino Acids Identified in Migraine Patients[END_REF]. However, other studies have shown that there is an increase in the level of TRP during the headache phase of the attack while a decrease is noted between the attacks (Deen et al. 2017;S. N. Young 2013). The fluctuations in the levels of TRP and its depletion are not a migraine trigger but rather an enhancer of the symptoms of migraine [START_REF] Carpenter | Tryptophan Depletion During Continuous CSF Sampling in Healthy Human Subjects[END_REF][START_REF] Van Der Stelt | Effects of Dietary Tryptophan Variations on Extracellular Serotonin in the Dorsal Hippocampus of Rats[END_REF][START_REF] Williams | Effects of Acute Tryptophan Depletion on Plasma and Cerebrospinal Fluid Tryptophan and 5-Hydroxyindoleacetic Acid in Normal Volunteers[END_REF]. Drummond was able to show that under Trp-depleted conditions, migraineurs suffer from an increase in headache and photophobia as well as an increase in nausea and dizziness (P. Drummond 2006). Adding to what he found, Jahromi et al, presented interesting data showing a 54-60% decrease in the risk of a migraine attack due to a daily dietary intake of TRP (Razeghi Jahromi, Togha, et al.

2019

). The entirety of the data obtained from the clinical studies opens the door that since TRP is the precursor of multiple compounds possibly involved in the pathogenesis of migraine (serotonin, kynurenine, and indoles), this could explain the link between TRP and migraine pathogenesis.

The Role of the Serotoninergic Pathway in Migraine

Serotonin is a vasoconstrictor located in the blood with a potential role in the modulation of nociception [START_REF] Taylor | Neurochemical Characterization of Extracellular Serotonin in the Rostral Ventromedial Medulla and Its Modulation by Noxious Stimuli[END_REF]. It directly activates the nociceptive afferents in order to augment the number of nociceptive impulses that are sent to the spinal cord. Its receptors are found within locations in migraine-related regions such as the thalamus, cortex, striatum, raphe nuclei, and cerebellum [START_REF] Barnes | A Review of Central 5-HT Receptors and Their Function[END_REF][START_REF] Varnäs | Autoradiographic Distribution of Serotonin Transporters and Receptor Subtypes in Human Brain[END_REF]. The important role of serotonin in migraine came about when Sicuteri et al., in addition to other researchers after found that the amount of 5-HIAA increased in the urine during the migraine attack, while the platelet concentration of serotonin decreased [START_REF] Curran | Total Plasma Serotonin, 5-Hydroxyindoleacetic Acid and p-Hydroxy-m-Methoxymandelic Acid Excretion in Normal and Migrainous Subjects[END_REF][START_REF] Sicuteri | Biochemical Investigations in Headache: Increase in the Hydroxyindoleacetic Acid Excretion during Migraine Attacks[END_REF].

In addition, reports of low levels of serum serotonin were found in migraineurs which were consistent with prior data [START_REF] Ren | Low Levels of Serum Serotonin and Amino Acids Identified in Migraine Patients[END_REF]. Also, in the case of reserpine-induced headaches, the infusion of serotonin was able to inhibit the spontaneous attack [START_REF] Kimball | Effect of Serotonin in Migraine Patients[END_REF].

The serotonin transporter (5-HTT) occurring mainly at the level of raphe nuclei and serotonergic projection areas such as the thalamus, uptakes the serotonin located in the synaptic cleft to reduce the effect of serotonin through a process controlled by a Na+/Cl-ion gradient [START_REF] Lesch | Regional Brain Expression of Serotonin Transporter MRNA and Its Regulation by Reuptake Inhibiting Antidepressants[END_REF][START_REF] Murphy | Serotoninergic Function in Neuropsychiatric Disorders[END_REF]). Interestingly, the distribution of 5-HTT is increased in migraine patients at the level of the brain stem [START_REF] Schuh-Hofer | Increased Serotonin Transporter Availability in the Brainstem of Migraineurs[END_REF]. Not only that but in the case of FHM, there exists a low level of serotonin in platelets in addition to a low transport capacity of 5-HTT coupled with reduced cerebrospinal fluid metabolite levels [START_REF] Horvath | Hemiplegic Migraine, Seizures, Progressive Spastic Paraparesis, Mood Disorder, and Coma in Siblings with Low Systemic Serotonin[END_REF]).

In addition to the transporter, serotonin receptors play a distinguished role in migraine. It is shown that the neurons of the trigeminal ganglia and the dorsal raphe are serotonergic (Berman et al. 2006;[START_REF] Lambert | The Lack of Peripheral Pathology in Migraine Headache[END_REF]. Moreover, the 5HT1B and 5HT1D

subtypes, where triptans act, are located at the level of trigeminal neurons, with their mRNA and protein detections in the trigeminal ganglia (J. C. A. [START_REF] Edvinsson | Lasmiditan and 5-Hydroxytryptamine in the Rat Trigeminal System; Expression, Release and Interactions with 5-HT1 Receptors[END_REF].

They are also located at the level of the axon of presynaptic neurons in the basal ganglia and frontal cortex. Furthermore, they are colocalized with the pain molecules, substance P, CGRP, and nNOS [START_REF] Hou | 5-HT1B and 5-HT1D Receptors in the Human Trigeminal Ganglion: Co-Localization with Calcitonin Gene-Related Peptide, Substance P and Nitric Oxide Synthase[END_REF]. Interestingly, the acute depletion of TRP in healthy human subjects (using seventy capsules of an amino acid mixture that doesn't contain TRP), induced changes in the expression and availability of 5HT1B in the cortex and raphe nuclei indicating a major link between TRP depletion and serotonin (Baldassarri et al. 2020).

One of serotonin's derivatives, melatonin, also has a role in migraine, where it can be an advantage for migraine through antagonizing glutamate-induced excitotoxicity, blocking NO synthesis, inhibiting the release of dopamine, and other functions (M. F. P. [START_REF] Peres | Potential Therapeutic Use of Melatonin in Migraine and Other Headache Disorders[END_REF].

The Role of the Kynurenine Pathway in Migraine

The contribution of kynurenine as a therapeutic potential in migraine came from the idea that KYNA is a competitive antagonist of the NMDA receptors that when inhibited protects against excitotoxicity induced by glutamate [START_REF] Kessler | A Glycine Site Associated with N-Methyl-d-Aspartic Acid Receptors: Characterization and Identification of a New Class of Antagonists[END_REF]).

Curto et al. observed a decrease in the levels of KYNA in addition to other metabolites in the serum of migraineurs suffering from chronic migraine compared to their controls in addition to having higher concentrations of TRP [START_REF] Curto | Altered Kynurenine Pathway Metabolites in Serum of Chronic Migraine Patients[END_REF]. The reduction of KYNA during migraine supports the theory of NMDA hyperactivity since KYNA is an antagonist of NMDA [START_REF] Hoffmann | Glutamate and Its Receptors as Therapeutic Targets for Migraine[END_REF]). Kynurenine's involvement was also shown in a model of CSD whereby the intake of kynurenine inhibited CSD probably through the increase of KYNA in the cortex [START_REF] Chauvel | Effect of Systemic Kynurenine on Cortical Spreading Depression and Its Modulation by Sex Hormones in Rat[END_REF]. From what has been found so far there is a positive link between migraine, TRP, kynurenine, glutamate, and NMDA that could explain the contribution of this pathway in the pathophysiology of migraine. Kynurenine metabolites also modulate migraine-related regions such as the LC. The administration of quinolinic acid intracerebroventricularly increased the activity of LC neurons, while the administration of KYNA inhibited the noxious stimulation evoked by central noradrenergic neurons in the LC (S. B. [START_REF] Abbott | C1 Neurons Excite Locus Coeruleus and A5 Noradrenergic Neurons along with Sympathetic Outflow in Rats[END_REF][START_REF] Ping | Modulation of Neuronal Activity of Locus Coeruleus in Rats Induced by Excitatory Amino Acids[END_REF]. A recent study shows that the depletion of TRP in cell culture increased the expression of AhR [START_REF] Solvay | Tryptophan Depletion Sensitizes the AHR Pathway by Increasing AHR Expression and GCN2/LAT1-Mediated Kynurenine Uptake, and Potentiates Induction of Regulatory T Lymphocytes[END_REF]). In addition, they found kynurenine, an endogenous ligand of AhR, whose cellular uptake was also increased through the augmentation of the expression of Lat1. As we know kynurenine is the metabolite of TRP, and TRP is involved in migraine pathogenesis (P. Drummond 2006). All of this adds to the activation of AhR by kynurenine under TRP-depleted conditions and potentially a link between TRP, AhRs, and migraine.

The Role of the Indole Pathway in Migraine

Indole and some of its downstream metabolites tryptamine and ILA have a role in the attenuation of inflammation at the level of the macrophages and intestinal epithelial cells [START_REF] Ehrlich | Indole-3-Lactic Acid Associated with Bifidobacterium-Dominated Microbiota Significantly Decreases Inflammation in Intestinal Epithelial Cells[END_REF]). In addition, indole, tryptamine, and IPA exert a plethora of effects by either activating or inhibiting the intestinal epithelial AhRs.

Furthermore, ILA and IPA are scavengers of free radicals and reducers of oxidative stress in systemic tissue [START_REF] Romani | Microbiota Control of a Tryptophan-AhR Pathway in Disease Tolerance to Fungi[END_REF]. IPA also exerts a neuroprotective effect and prevents weight gain in animal models and is an inhibitor of gut dysbiosis [START_REF] Konopelski | Indole-3-Propionic Acid, a Tryptophan-Derived Bacterial Metabolite, Reduces Weight Gain in Rats[END_REF]Z.-H. Zhao et al. 2019).

Indoles have shown a contribution in nociception whereby through the direct activation of transient receptor potential ankyrin 1 (TRPA1) nociceptors, they induce a painful response and a role in pain signaling [START_REF] Chung | Common Bacterial Metabolite Indole Directly Activates Nociceptive Neuron through Transient Receptor Potential Ankyrin 1 Channel[END_REF]). However, currently, there are no concrete links between indoles and migraine. The actual mechanism by which indoles contribute to migraine pathogenesis is quite vague. One study measured the levels of tryptamine in the plasma of chronic migraineurs, which are lower than their respective controls [START_REF] D'andrea | Tryptamine Levels Are Low in Plasma of Chronic Migraine and Chronic Tension-Type Headache[END_REF]). As a function of time, more work should be done to further understand the contribution of this pathway to migraine.

Migraine is a neurobiological disorder with episodic manifestations. However, in approximately 3% of patients, these episodic patterns take on a chronic form.

Although the mechanisms of this progression are unclear, the neuroinflammatory state could play a key role. During headaches, migraineurs report cutaneous cephalic hypersensitivity (correlated with central sensitization) and dysfunctional descending pain modulatory pathways (at the level of the brainstem among others). These neuronal alterations are concomitant with increased plasmatic levels of pro-inflammatory cytokines during and between attacks. Moreover, migraine is comorbid with other chronic inflammatory disorders, and it has been described that under nociceptive conditions, glial cell function is dysregulated. Hence, we first hypothesized that migraine is coupled with an enhanced inflammatory response, which modulates both the central sensitization and the brainstem function, contributing to the progression of migraine toward a chronic state. The inflammatory signaling can be initiated by TRPderived metabolites through the activation of AhR. Both players have already been involved in comorbid inflammatory diseases, and TRP metabolism is impaired in migraineurs. Therefore, our second hypothesis came that the dysregulation of TRP metabolism and AhR activity promote an abnormal neuroinflammatory state during migraine that contributes to disease progression (Figure 21).

Thus, we studied the involvement of TRP and its metabolites in the inflammatory-related processes during migraine initiation and chronification, and in the therapeutic efficacy of triptans. We chronically dysregulated the metabolism of TRP by dietary TRP depletion and examined the potential neuronal changes at the level of the LC and the Sp5C in an experimental mouse model of migraine-like pain induced by systemic ISDN administration. We focused our study on these two regions for several reasons: (1) the Sp5C is a key player in the nociceptive integration at the level of the trigeminovascular system, and its activation contributes to central sensitization, and hence, to cutaneous hypersensitivity; (2) the LC has been clinically and preclinically implicated in migraine, it modulates the trigeminal system by its descending noradrenergic inputs, and it is involved in pain chronification. Our experimental approach combined behavioral assessment (pain, light aversion, and anxiety), immunohistochemistry, and biomolecular techniques (RT-PCR, HPLC, western blot, and ELISA). To address our hypotheses, this project was mainly divided into two studies, with some complementary results. The specific objectives for each study were:

.

Study I:

To investigate the consequences of TRP dysregulation on:

the onset of migraine-like pain induced by a single ISDN administration in male and female mice through the assessment of the two most prevalent sensory symptoms: cephalic mechanical hypersensitivity and light hypersensitivity.

the effectiveness of sumatriptan to block cephalic mechanical and light hypersensitivities induced by ISDN administration in both sexes.

.

Study II:

To investigate the consequences of TRP dysregulation on:

the migraine progression after repeated ISDN administration in female mice, through the behavioral assessment of cephalic mechanical sensitivity, and the evaluation of the LC function.

the systemic (plasmatic) and local (in the LC and the Sp5C) inflammatory state in female mice after repeated ISDN administration.

To characterize the contribution of AhR on migraine progression through behavioral assessment of cephalic mechanical sensitivity after AhR agonist/antagonist drug administration, and their activity at the level of the LC and the Sp5C after repeated ISDN administrations.

INTRODUCTION

Migraine is a prevalent incapacitating neurological disorder ranked as the third most common disease in the world (Vos et al. 2016). It is characterized by unilateral, pulsating headache attacks, accompanied by sensory disturbances, such as cephalic mechanical (CMH) and light (LH) hypersensitivities. Sumatriptan, a 5hydroxytryptamine (5-HT) 1B/1D receptor (5-HTR1B/1D) agonist, is recommended as a first-line abortive drug for patients suffering from moderate to severe migraine attacks.

Nevertheless, ∼30% of migraine patients are non-responders to sumatriptan, and individual responsiveness to triptans is variable (J.-W. Wu et al. 2022). To date, the response variability is not fully understood, and only a few studies have identified the predictors for triptan response in migraine. Current evidence showed that a lower pain severity prior to treatment and a higher polygenic risk score was associated with a better response to triptans (Diener et al. 2004;Kogelman et al. 2019). An early study suggested that triptan efficacy is less optimal after a patient develops cutaneous Evidence supports 5-HT transmission as a main factor involved in migraine pathogenesis. Migraineurs present decreased 5-HT levels between attacks and increased levels during the ictal period (Deen et al. 2017). Moreover, changes in the sensitivity to 5-HT agonists (triptans) might occur during attacks probably due to a defect in 5-HT metabolism (Deen et al. 2018;Proietti-Cecchini, Afra, and Schoenen 1997;Sakai et al. 2008). The changes in 5-HT turnover or the sensitivity of its receptors might participate in the disturbances of sensory processes (Deen et al. 2017). Thus, it is proposed that migraine headaches might develop during a period of attenuation in 5-HT activity and consequent stimulation of 5-HT receptors (Gupta, Nahas, and Peterlin 2011).

Tryptophan (TRP) is an essential precursor required for different metabolic reactions

MATERIALS AND METHODS

Animals

Adult male and female C57BL/6 mice (7 to 12 weeks old) were obtained from Charles River (L'Arbresle, France) or born in our amenities. Mice were housed under a 12 h light/dark cycle with ad libitum access to food and water. Environmental enrichment was provided for nest building and shelter and transferred with mice when cages were cleaned. Procedures were conducted according to the experimental design (Figure 1A)

during the dark phase (between 8 AM and 3 PM). At the end of the experiments, mice were sacrificed using a lethal dose of sodium pentobarbital (150 mg/kg, i.p., CEVA Santé Animale, Libourne, France). Death was confirmed by permanent cessation of cardiorespiratory functions.

All efforts were made to minimize the number of animals used. A total of 224 animals were used to complete this study: 87 for the von Frey test, including weight and food consumption assessments; 72 for the modified elevated plus maze (mEPM) test; 40 for ELISA and HPLC; and 25 for the western blots.

Study Approval

All procedures and analyses were performed following ARRIVE (Animals in Research:

Dietary Tryptophan Depletion

Mice were randomly assigned to one of two dietary conditions: the control diet (balanced diet, BAL) contained 2.3 g of TRP per kg of food, which is considered a normal intake in mice (John and Bell 1976); or the TRP-deficient diet (TRP -) contained 0.3 g/Kg of TRP. Both diets were manufactured by SAFE ® Custom Diets company (Augy, France) and formulated as described in Table 1, with the required levels of amino acids necessary for optimal health. The form of the pellets was similar between both diets, with a 10-12 mm diameter and a crushing resistance of ~5 kgf/cm 2 . The body weight of mice (Figures S1A andS1A2) and their food consumption (Figures S1B1 andS1B2) were measured on a daily basis as an indication of the well-being of animals.

Drug administration

A single intraperitoneal (i.p.) injection of ISDN (Risordan®, Sanofi-Aventis, Paris, France; 10 mg/kg in a 10 ml/kg volume) or vehicle (0.9% NaCl) was administrated as previously described (Dallel, Descheemaeker, and Luccarini 2018). Sumatriptan succinate (S1198, Sigma-Aldrich, St. Quentin Fallavier, France) was injected i.p. ( 1mg/kg in 10 ml/kg volume, dissolved in saline) 5 min after ISDN/vehicle injection.

Behavioral Tests

Briefly, mice were first allowed to acclimatize to the room used for behavioral testing as well as to innocuous mechanical stimulation for 3 days before the start of the behavioral sensory testing. Test sessions took place during the light phase, between 11 a.m. and 7 p.m.

Cephalic Mechanical Sensitivity

Periorbital sensitivity to non-nociceptive mechanical stimuli was measured following the modified up-down method described previously (Alba-Delgado et al. 2018). A series of eight von Frey filaments (0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, and 1.0 g; 1.0 g cut-off; Bioseb, Vitrolles, France) were presented perpendicularly to the left periorbital surface of the forehead. Testing began with the 0.4 g filament. After drug administration, the last weak von Frey filament was used. The stimuli were presented using an up-down sequence five times at 5 s intervals. Detection of a positive response (eye winking, turning the head away, attacking filament, and/or body freezing) three or more times lead to choosing the next weaker stimulus. In the absence of a positive response, a stronger stimulus was presented. The cephalic mechanical sensitivity threshold (in g) was determined as the weakest filament used which obtained three or more positive responses. The cephalic sensitivity threshold was measured before (baseline) and every hour for 3 h after ISDN/vehicle administration. Analysis exclusion was done for mice showing distress signs after drug injection, or those that did not habituate to the 0.6 g filament before the testing day.

Light Sensitivity

Light avoidance behavior was evaluated in a modified version of the elevated plus maze test (mEPM) according to (Chanda et al. 2013). An LED lighting system (5000 K;

Greenice SA, Toledo, Spain) was placed on top of the closed compartments (termed "light sides") while the open compartments (termed "dark sides") were not modified.

The left closed arm was set at 300 lux while the right closed arm was set at 50 lux (the brightness was checked with a lux meter at the level of the mouse's eye). The test lasted 10 min (Figure 1B): during the first 5 min (LED-Off condition), the whole device was in darkness (<20 lux); then, only the light sides (closed arms) were lit for 5 min (LED-On condition). The animal's free exploratory behavior was filmed by an infrared camera connected to a video tracking analysis software (EthoVision XT v13.0; Noldus Information Tech., Wageningen, Netherlands). The light sensitivity threshold was calculated as a light aversion index (LAI) (Matynia et al. 2012). Mice with negative scores or around zero were considered to have no light avoidance. Positive scores were considered to be light aversive. The percentage of time spent in the closed arms during the LED-Off condition was used to evaluate the anxiety-like behavior.

Blood and Tissue Preparation

Sample collection was performed 1 h after ISDN/vehicle injection (Figure 1A). Mice were fasted for 1 h and then anesthetized with an i.p. administration of 25% urethane (1.5 g/kg i.p.). Blood was collected after decapitation and stored in 2 ml tubes without anticoagulant at room temperature. After complete coagulation, samples were centrifuged at 15000 x g for 10 min at room temperature, and the serum was carefully collected. At the same time, the brain was removed from the skull and the two spinal trigeminal nucleus caudalis (Sp5c) were manually dissected. Blood and brain samples were immediately frozen in liquid nitrogen and then stored at -80 °C for later use.

Detection of Metabolites

TRP or 5-HT levels in serum were measured by using the Enzyme-Linked Immunosorbent Assay (ELISA) kits (BA E-2700 or BA E-8900 respectively, ImmuSmol, Bordeaux, France). Provider instructions were followed.

High-Performance Liquid Chromatography (HPLC) with electrochemical detection was used to quantify the levels of TRP in the Sp5C. Corresponding tissue was first homogenized using an Ultra-Turrax T8 homogenizer in 15 μl/mg homogenization buffer (0.1 M HClO4 and 100 μl EDTA). The crude homogenate was centrifuged at 21,000 x g for 15 min at 4 ºC, and the supernatant was diluted 1:5. Samples were then filtered by centrifugation at 15,000 x g for 15 min using Costar® Spin-X® Centrifuge Technologies, Madrid, Spain). TRP amperometric detection was carried out using a Hewlett-Packard model 1049A detector (Agilent Technologies Deutschland GmbH, Waldbronn, Germany) at an oxidizing potential of +950 mV.

Western Blot

The Sp5C samples were homogenized using a lysis buffer containing EDTA (0.5 M),

Trizma base (50 mM), sodium chloride (10 mM), Triton 100% (0.25%), and supplemented with a cocktail of protease and phosphatase inhibitors (Thermo Fisher Scientific, Illkirch-Graffenstaden, France). An equal amount of total protein (20 µg/each sample) was resolved in each lane of 10% SDS-PAGE gels using Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad Laboratories) at 80 V for 15 min followed by 200V for approximately 30 min. The resolved proteins were then transferred onto a PVDF membrane (Merck Chimie SAS Île-de-France, France) using a Mini Trans-Blot® Cell (Bio-Rad Laboratories) at 100 mA for 1 h. Thereafter, membranes were incubated at 25 °C for 1 h with a solution containing 1% Casein in tris-buffered saline (TBS) to block non-specific bindings. Membranes were then incubated overnight at 4 °C with the primary antibodies against 5-HTR1B or 5-HTR1D (1:500; OSS00185W or OSS00182W respectively, Osenses, Keswick, Australia). Subsequently, they were washed with TBS 1X and incubated with the StarBright Blue 700 goat anti-rabbit secondary antibody

(1:2,500; 64297093, Bio-rad Laboratories, Marnes-la-Coquette, France) at room temperature for 2 h. Blots were visualized with the ChemiDoc XRS system (Bio-Rad Laboratories) and analyzed using the software Image Lab (v6.1, Bio-Rad Laboratories).

Statistical Analysis

Data are presented as mean ± SEM. The number (n) of mice utilized for each analysis was shown in Figures or Tables. 2, and in Table S1. The level of significance was set to p < 0.05. All statistical analyses and graphs were performed using GraphPad Prism (v8.4.0, GraphPad Inc., La Jolla, CA, USA). Figures

were made using CorelDraw Graphics software (v12.0, Ottawa, Canada).

RESULTS

TRP deficiency reduces serum TRP and 5-HT concentrations in both sexes

First, we examined the serum concentration of TRP in both male and female mice subjected to one week of a balanced (BAL) or TRP-deficient diet (TRP -) (Table 3). In the BAL diet group, vehicle-injected females showed significantly higher TRP levels (176.0 ± 16.0 µM) than vehicle-injected males (90.2 ± 10.9 µM). After dietary TRP depletion, the serum TRP concentration decreased by 68.5 % and 66.7 % in female and male mice, respectively (Table 3). No differences were found between the sexes (Table 3).

The basal serum concentration of 5-HT was similar in both sexes in the BAL diet group (1.63 ± 0.05 µM vs. 1.68 ± 0.08 µg/ml respectively; Table 3). After dietary TRP depletion, the serum 5-HT concentrations decreased by 12.9 % in vehicle-injected females (1.42 ± 0.04 µM) and 22 % in males (1.31 ± 0.03 µM) (Table 3). ISDN-injected males under a TRP -diet did not show reduced 5-HT levels (1.56 ± 0.04 µM), which were higher than vehicle-injected males (1.31 ± 0.03 µM) and similar to groups under a BAL diet (Table 3).

TRP deficiency worsens ISDN-induced CMH in both sexes

Basal values were similar in BAL (female: 0.93 ± 0.07 g, male: 0.93 ± 0.07g) and TRP - diet groups (female: 0.87 ± 0.08 g, male: 0.93 ± 0.07 g) of both sexes (Figures 2A1 and2A2).

A single ISDN administration induced CMH in both sexes, peaking at 1 h and lasting for at least 3 h in the BAL diet group (Figures 2A1 and2A2). Time-course of CMH was similar between sexes (female AUC: 0.70 ± 0.13 AU vs. male AUC: 0.65 ± 0.16 AU;

Figure 2B). Interestingly, under TRP-deficient conditions, ISDN also induced CMH with a similar peak at 1 h, but with a significant lack of recovery at 3 h (females: 0.04 ± 0.02 g; males: 0.10 ± 0.03 g) compared to their BAL counterparts (females: 0.48 ± 0.04 g; males: 0.45 ± 0.13 g; Figures 2A1 and2A2). Indeed, the AUC measured between 1 to 3 h post-injection confirmed these significant differences between both diets, in females and males (Figure 2B). No differences were observed between the sexes.

TRP deficiency blocks the efficacy of sumatriptan to prevent the ISDN-induced CMH

In the BAL diet group, sumatriptan (1 mg/kg i.p.) delivered 5 min after ISDN was able to inhibit ISDN-induced CMH in both sexes (Figures 2C1 and2C2). The therapeutic efficacy of sumatriptan was similar between the sexes, as shown by their similar AUC (female: 1.22 ± 0.22 AU, male: 1.44 ± 0.24 AU; Figure 2D).

After dietary TRP deficiency, sumatriptan did not affect ISDN-induced CMH in both sexes (Figures 2C1 and2C2). One hour post-administration, the cephalic mechanical thresholds were not different between the vehicle (females: 0.02 ± 0.01 g; males: 0.11 ± 0.07 g) and sumatriptan (females: 0.12 ± 0.07 g; males: 0.01 ± 0.01 g) injected animals.

This was confirmed by the significant differences in the AUC between TRP -ISDN groups (females: 0.22 ± 0.14 g; males: 0.03 ± 0.01 g) compared to BAL groups (females: 1.22 ± 0.22 g; males: 1.44 ± 0.24 g; Figure 2D).

ISDN induces LH exclusively in females, which is not altered by TRP deficiency

In the BAL diet group, vehicle-injected female mice did not show LH neither at 50 lux nor 300 lux, as shown by the negative LAI (-0.20 ± 0.15 and -0.27 ± 0.13 respectively;

Figure 3A). However, vehicle-injected males show a positive LAI at 300 lux (0.29 ± 0.12; Figure 3B), indicating an innate LH. ISDN administration evoked a positive LAI in females at 300 lux (0.35 ± 0.08; Figure 3A) but did not modify the innate LH in males (0.32 ± 0.10; Figure 3B). TRP deficiency did not affect light sensitivity neither in vehicle-injected nor in ISDN-injected female or male mice, at both 50 lux and 300 lux (Figures 3A and3B).

To verify that LH measured in the mEPM was not linked to anxiety, the time spent in the closed arms during the LED-Off condition was measured (Figure S2). Animals did not present anxiety-like behavior neither after BAL or TRP -diets, nor after the ISDN injection.

TRP deficiency blocks the efficacy of sumatriptan to prevent the ISDN-induced

LH

In the BAL diet group, sumatriptan prevents ISDN-induced LH in female mice in the mEPM (Figure 3B), as shown by the negative LAI at 300 lux in the sumatriptan-treated ISDN group (-0.14 ± 0.07). Interestingly, in the TRP -diet group, the efficacy of sumatriptan was affected by the TRP deficiency. ISDN-injected females showed a positive LAI at 300 lux (0.36 ± 0.08), similar to ISDN-injected mice (0.54 ± 0.08; Figure 3A). Sumatriptan did not affect the innate LH in males.

TRP deficiency does not change the TRP concentration in the Sp5C

To evaluate if the sumatriptan inefficacy was linked to the dysregulation of TRP metabolism in the Sp5C following one week of a TRP-deficient diet, we quantified the TRP concentration using HPLC in this region (Table 3). No differences between sexes were observed, hence the data were pooled to increase the power of the statistical analysis. Surprisingly, the TRP-deficient diet did not reduce TRP levels (2.7 ± 0.5 nM)

which were similar to the BAL condition (3.0 ± 0.3 nM). Systemic ISDN administration also did not modify TRP levels in the SP5C (at 1 h post-injection), in neither BAL nor TRP-deficient diets.

TRP deficiency increases the expression of 5-HTR1D in the Sp5C

We assessed the expression of the 5HT1D and 5HT1B receptors in the Sp5C through western blot. Since no difference was observed in the 5HT1D and 5HT1B receptors expression between males and females, we pooled the data. In the BAL diet group, ISDN does not affect the 5HT1D and 5HT1B receptor expression in the Sp5C. Dietary TRP deficiency alone also did not induce changes in the 5HT1D and 5HT1B receptors in the Sp5C (Figures 4A2 and4B2). Interestingly, ISDN administration in the TRP -diet group produced a dramatic increase of 5HTR1D expression (Figure 4A2), but not 5HTR1B in the Sp5C (Figure 4B2), compared to vehicle-injected mice under the same diet.

DISCUSSION

The present study demonstrated that chronic dietary TRP depletion reduces the availability of plasmatic TRP which (i) worsens ISDN-induced CMH in both sexes and

(ii) reduces the efficacy of sumatriptan on both ISDN-induced CMH and LH. These changes were associated with increased 5-HTR1D expression in the Sp5C and reduced serum levels of 5-HT.

ISDN as a preclinical model of migraine

The headache research field has well-established animal models that improved our understanding of the peripheral and central headache mechanisms and their interaction.

Several models exist simulating different aspects of migraine with certain limitations (Harriott et al. 2019). Here, we used ISDN as an animal model of migraine because it reliably produces headaches in migraineurs, though less often in healthy subjects (Bellantonio et al. 1997;Castellano et al. 1998), and results in CMH associated with sensitization of the trigemino-cervical complex in rats (Dallel, Descheemaeker, and Luccarini 2018;Flores Ramos et al. 2017). Moreover, sumatriptan and olcegepant, two specific acute antimigraine drugs, block ISDN-induced CMH (Dallel, Descheemaeker, and Luccarini 2018). As in rats, we found that a single ISDN injection induced reversible CMH that was similar in mice in both sexes.

Moreover, ISDN evoked LH exclusively in females. LH is the most bothersome symptom associated with a migraine attack but is also reported in headache-free periods in patients (Pinheiro et al. 2021). Light aversive behavior has also been previously reported after an acute NTG injection, in both male (Markovics et al. 2012) and female mice (Eller et al. 2021). However, no differences were detected after NTG (Targowska-Duda et al. 2020) or CGRP administrations (Mason et al. 2017). But, our results were in accordance with clinical studies that reported a higher incidence of LH during migraine attacks in women than men (Boardman et al. 2003;Bolay et al. 2015). The lack of LH in males could be attributed to the fact that females are more sensitive to nitric oxide (NO) donors than males, as shown by the higher activity of the NO synthase in females compared to males (Oydanich et al. 2019). Moreover, males showed a higher innate sensibility to light than females, which could support different behavioral and neuronal mechanisms for light adaptation (Chellappa 2021).

Both CMH and LH were blocked by systemic sumatriptan administration, which was consistent with previous studies in ISDN-injected rats (Dallel, Descheemaeker, and Luccarini 2018) and after an acute NTG administration in rats [START_REF] Farajdokht | Chronic Ghrelin Treatment Reduced Photophobia and Anxiety-like Behaviors in Nitroglycerin-Induced Migraine: Role of Pituitary Adenylate Cyclase-Activating Polypeptide[END_REF] and mice (Casili et al. 2020).

Dietary deficit in TRP dysregulates systemic TRP metabolism and prolongs cephalic pain

Prior to any change in the availability of TRP, we saw a higher level of baseline TRP in females than in males which adds up to the higher circulating levels of TRP in females compared to males in animal studies (Jans, Lieben, and Blokland 2007). Here, we used a TRP-deficient diet for one week before inducing migraine-like pain, which reduced serum TRP and 5-HT levels in male and female mice. Following the implementation of a one-week TRP-deficient diet a 70% decrease in the serum levels of TRP was attained following one week coupled with a significant decrease in its metabolite serotonin.

During this decrease, we have shown no sign of a deterioration in the well-being of the animals represented by no robust decrease in their weights, change in the amount of food consumed, and no anxiogenic behavior. This decrease has been priorly shown in rats on low TRP-containing diets for periods of up to one week (Franklin, Cowen, and Craven 1995) and in humans on low-calorie TRP-restricting diets for three weeks [START_REF] Walsh | Dieting Decreases Plasma Tryptophan and Increases the Prolactin Response to D-Fenfluramine in Women but Not Men[END_REF] The testing period in the mEPM was divided into two parts: a 5 min LED-Off period (with the LED system turned off) followed by a 5 min LED-On (with the LED system turned on in the closed arms). 123

STUDY II INTRODUCTION

Chronic migraine (CM) is a disabling neurological condition with a global prevalence of 2%, 2.5 to 6.5-fold higher in women than in men (Natoli et al. 2010). It is characterized by recurrent headache attacks (≥15 days/month) (IHS 2018), accompanied by cutaneous hypersensitivity in more than 40% of cases (Louter et al. 2013). Each year, ~3% of episodic migraineurs suffer from increasing attack frequencies and progress gradually to a CM state (A. I. Scher et al. 2003). Although underlying physiological mechanisms of this progression remain unclear, altered modulation of pain pathways and trigeminal central sensitization represent physiological correlates of chronification [START_REF] Bigal | Concepts and Mechanisms of Migraine Chronification[END_REF].

Impaired descending inhibition from the brainstem is a contributor to this progression (Akerman, Holland, and Goadsby 2011). Among the brainstem nuclei, the locus coeruleus (LC) has been involved in the pathophysiology of migraine. It is activated by intracranial trigeminal stimulation in rats (Ter Horst et al. 2001). As the main source of noradrenaline (NA) descending inputs to the trigeminal complex, it can modulate nociceptive responses (Sasa et al. 1974;Vila-Pueyo et al. 2019). In addition, neuroimaging studies highlighted altered LC functional connectivity in migraineurs (Moulton et al. 2014), and LC activation following nitroglycerin (NTG) infusion (Maniyar et al. 2014) and during spontaneous headaches (Weiller et al. 1995).

Emerging data also suggest that neuroinflammation is a modulator of central sensitization. Increased levels of pro-inflammatory chemokines and cytokines have been detected during ictal and interictal periods in migraine sufferers (Biscetti et al. 2022). In rats, NTG-induced CM was correlated with increased transcriptional activity of interleukin (IL)-6 and tumor necrosis factor (TNF)α in the trigeminal ganglia and pons (including the LC) (Greco et al. 2017), and with microglial activation in the spinal trigeminal nucleus caudalis (Sp5C), contributing to central sensitization (He et al. 2019).

Together, these data place both the LC and neuroinflammation as potential key players in migraine progression. However, how neuroinflammatory signaling is initiated and the exact role of the LC in this process are unknown. Recently, tryptophan (TRP) and its metabolites (mainly kynurenine -KYN-and serotonin derivatives) have been demonstrated to be associated with neuroinflammation (Rothhammer et al. 2016) and migraine pathophysiology (Deen et al. 2019;P. Drummond 2006;Körtési, Spekker, and Vécsei 2022). TRP regulates inflammatory processes through the activation of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor involved in neural and glial functions (Juricek and Coumoul 2018), and linked to various chronic diseases (Rothhammer and Quintana 2019). Although its contribution to migraine has not yet been studied, evidence supports it: (i) TRP-derived AhR agonists are key regulators of migraine pathophysiology (Deen et al. 2019;Körtési, Spekker, and Vécsei 2022); AhR is expressed in migraine-related-regions (Petersen et al. 2000) including the LC (E. Kimura et al. 2021); (ii) its activation directly regulates the tyrosine hydroxylase (TH) expression [START_REF] Akahoshi | Effect of Dioxins on Regulation of Tyrosine Hydroxylase Gene Expression by Aryl Hydrocarbon Receptor: A Neurotoxicology Study[END_REF], and hence the production of the analgesic NA; while its depletion increases cutaneous sensitivity in chronic neuropathic mice (Sheu et al. 2022).

Here, we evaluate the contribution of TRP metabolism and AhR activity in regulating migraine-like pain induced by the systemic isosorbide dinitrate (ISDN) administration (Dallel, Descheemaeker, and Luccarini 2018): indirectly by submitting female mice to a chronic TRP deficient diet (that reduces endogenous AhR agonists); and directly by pharmacological AhR activation/inhibition. We also investigated the neuroinflammatory state, including glial activation and cytokine release. Our findings establish a critical link between perturbed TRP metabolism, dysregulated neuroinflammation in the LC, and AhR in migraine progression.

MATERIAL AND METHODS

Animals

Adult female C57BL/6 mice (7 to 12 weeks old) were obtained from Charles River (L'Arbresle, France) or born in our amenities. Mice were housed in the same room under standard conditions (12h light/dark cycle, ad libitum access to food and water, environmental enrichment). Procedures were performed according to the experimental design (Figure 1A) between 8 AM and 3 PM (dark phase). At the end of the experiments, mice were sacrificed (150 mg/kg, i.p. of sodium pentobarbital; CEVA Santé Animale, Libourne, France) and death was confirmed by permanent cessation of cardiorespiratory functions. All efforts were made to minimize the number of animals used.

Study approval

Experiences

Diets

Mice were randomly assigned to one of two dietary conditions: the balanced diet (BAL; control diet) that contained 2.3 g of TRP per kg of food; or the TRP deficient diet (TRP -) that contained 0.3 g of TRP/kg. Diets were formulated with 60.4% of cereals, 14.0% of vegetal proteins, 4.8% of vitamins and minerals mixture, 10.0% of carbohydrates, 10.0% of oils and fats, and 0.9% of amino acid mixture, which contains (in g per kg of food): 13.0 arginine, 3.2 cystine, 34.1 glycine, 7.6 lysine, 4.1 methionine, and the corresponding amount of TRP (SAFE ® Custom Diets company, Augy, France).

Both diets have similar pellets (10-12 mm diameter) and crushing resistance (~5 kgf/cm 2 ). Each diet was started at 7-8 weeks of age and continued from 7 to 30 days according to the experimental design (Figure 1A). Body weight was measured on a daily basis during the observation period.

Drug administration

Single (ISDN-1 or Veh-1) or repeated (ISDN-3 or Veh-3; twice per day for two days) intraperitoneal (i.p.) injections of ISDN (10 mg/kg in a 10 ml/kg volume; Risordan®, Sanofi-Aventis, Paris, France) or vehicle (0.9% NaCl) were administrated as described in (Dallel, Descheemaeker, and Luccarini 2018). 6,2′,4′-trimethoxyflavone (TMF; France), an AhR agonist, was injected i.p. (10 mg/kg in 10 ml/kg volume in saline) 5 h before the single ISDN/vehicle injection.

Behavioral assessment

Cephalic mechanical sensitivity

The periorbital mechanical sensitivity to non-noxious stimuli was measured by using a series of eight von Frey filaments (from 0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, and 1.0 g; 1.0-g cut-off; Bioseb, Vitrolles, France) as described previously (Alba-Delgado et al.

2018

). For the first three days, habituation was done using the 0.6 g filament. Testing began with the 0.4 g filament for baseline measurement and with the last weak filament used for measurements after drug administration. Stimuli were presented perpendicularly to the left periorbital surface of the forehead, using an up-down sequence five times at intervals of at least 5 s. The detection of a positive response three or more times lead to choosing the next weaker stimulus. In the absence of a positive response, a stronger stimulus was presented. Eye winking, turning the head away, attacking the tip of the filament, and/or body freezing were considered positive detection responses. The cephalic mechanical sensitivity threshold (in g) was determined as the weakest von Frey filament used which obtained three or more positive responses.

Measurements were performed before (baseline), every hour for 3 h after single or repetitive ISDN/vehicle administrations, and prolonged to 30 days after the last repeated ISDN/vehicle injection (Figure 1A). The effect of ITE or TMF was also measured (Figure 1A). Mice showing distress signs not habituated to the 0.6 g filament before the testing day were excluded.

Extra-cephalic Mechanical Sensitivity

The mechanical sensitivity of the hind paw to non-noxious stimuli was also measured by von Frey filaments (from 0.008 to 1.4 g; 1.4 g cut-off; Bioseb, Vitrolles, France).

Habituation was done using the 1.0 g filament. Stimuli were presented perpendicularly to the bottom of the left or right hind paw, using the up-down sequence five times at intervals of at least 5 s. Like cephalic sensitivity, the hind-paw mechanical sensitivity threshold (in g) was determined as the weakest von Frey filament used which obtained three or more positive responses. Paw shaking or withdrawal, attacking the filament tip, and/or freezing were considered positive responses. As no differences between the left and right paw were found, an average of values was done. Measurements were performed before (baseline), every hour for 3 h after repetitive ISDN/vehicle administrations, and prolonged to 3 days after the last injection, according to the experimental protocol (Figure 1A).

Anxiety-like behavior

The assessment of anxiety-like behavior was done using the open-field test (OFT). The free movement of mice was recorded in an open square box (50x50x30 cm high) for a 5-min duration using a digital infrared camera (DMK 21AF04, The Imaging Source Europe GmbH, Bremen, Germany) connected to an analysis software (EthoVision XT, version 13.0; Noldus Information Technology, Wageningen, Netherlands). Two virtual zones were delimited: center (in the middle of the arena; 30x30 cm) and periphery (surrounding border zone with 20 cm in width). The time spent in the peripheral zone (in %) and the total distance covered in the open area (in m) were measured 30 min after the last repeated ISDN/vehicle injection and 30 days after the start of the diet (Figure 1A).

Quantification of Metabolites and Cytokine Levels

Blood and tissue preparation

Sample collection was performed 1 h after ISDN/vehicle injection (Figure 1A). Mice were fasted for 1 h and then anesthetized with 25% urethane (1.5 g/kg i.p.). Blood was collected after decapitation in tubes with cold heparin (50 µl/tube; Heparine Choay, Vidal, France) and centrifuged at 5000 x g for 12 min at 4 °C. Plasma was carefully collected and immediately frozen in liquid nitrogen. At the same time, the brain was also collected, and the two LC manually dissected and immediately frozen in liquid nitrogen. All samples were finally stored at -80 °C for later use.

Detection by HPLC

The quantification of plasmatic and cerebral TRP, KYN, and NA concentrations was performed by high-performance liquid chromatography (HPLC) with electrochemical detection according to (Unzueta-Larrinaga et al. 2023). Samples were first homogenized using an Ultra-Turrax T8 homogenizer in 15 μL/mg homogenization buffer (0.1 M HClO4 and 100 μl EDTA). The crude homogenate was centrifuged at 21,000 x g for 15 min at 4 ºC, and the supernatant was diluted 1:5. All samples were then filtered by centrifugation at 15,000 x g for 15 min using Costar® Spin-X® Centrifuge Tubes (0.22 μm Pore CA Membrane, Merck Life Science S.L.U., Madrid, Spain). The concentration of TRP, KYN, and NA was estimated with reference to standards prepared and injected on the same day. The mobile phase composition was 150 mM H2NaPO4, 0.2 mM EDTA, 4.3 mM octyl sodium sulfate (pH 6.3), and 8% (vol/vol) methanol. The filtered mobile phase was delivered at a flow rate of 0.2 ml/min by a Hewlett-Packard model 1200 pump (Agilent Technologies Deutschland GmbH, Waldbronn, Germany). Sample separation was carried out at 30 °C on a Zorbax Eclipse Plus column (3.5 μm C18, 2.1×150 mm, Agilent Technologies, Madrid, Spain). Metabolites' amperometric detection was carried out using a Hewlett-Packard model 1049 A detector (Agilent Technologies Deutschland GmbH, Waldbronn, Germany) at an oxidizing potential of + 950 mV.

Detection by ELISA

The quantification of the plasmatic concentration of pro-inflammatory cytokines (IL-1β, IL-6, interferon-gamma -IFNγ-and TNFα) was performed by a Multiplex ELISA kit (ARG82842, ArigoPLEX®, Arigo Biolaboratories, Hsinchu City, Taiwan) according to the manufacturer's protocol. The intensity of sample color was measured at 450 nm by a microplate reader (BioTek Epoch Microplate Spectrophotometer, BioTek Instruments SAS, Colmar, France). The final concentration of cytokines was determined through comparisons with the standard curve.

Quantification of Protein and mRNA Relative Expression

Tissue preparation

Sample collection was performed 1 h after ISDN/vehicle injection (Figure 1A). Mice were fasted for 1 h and then anesthetized with 25% urethane (1.5 g/kg i.p.). Brains were collected after decapitation, and both right and left LC were manually dissected and immediately frozen together in liquid nitrogen. All samples were finally stored at -80 °C for later use.

Western blot

LC samples were homogenized using a lysis buffer containing EDTA (0.5 M), Trizma base (50 mM), sodium chloride (10 mM), Triton 100% (0.25%), and supplemented with a cocktail of protease and phosphatase inhibitors (Thermo Fisher Scientific, France). An equal amount of total protein (20 µg/each sample) was resolved in each lane of 10% SDS-PAGE gels using Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad Laboratories) at 80 V for 15 min followed by 200V for approximately 30 min. The resolved proteins were then transferred onto a PVDF membrane (Merck Chimie SAS Île-de-France, France) using a Mini Trans-Blot® Cell (Bio-Rad Laboratories) at 100 mA for 1 h. Thereafter, membranes were incubated at 25 °C for 1 h with a solution containing 1% Casein in tris-buffered saline (TBS) to block non-specific bindings.

Membranes were then incubated overnight at 4 °C with the primary antibody rabbit anti-TH (1:1000; ab152, Merck Millipore SAS, Île-de-France, France). Subsequently, they were washed with TBS 1X and incubated with the StarBright Blue 700 goat antirabbit secondary antibody (1:2,500; 1200416, Bio-rad Laboratories, Marnes-la-Coquette, France) at room temperature for 2 h. Blots were visualized with the ChemiDoc XRS system (Bio-Rad Laboratories) and analyzed using the software Image Lab (v6.1, Bio-Rad Laboratories).

.

Quantification of Immunolabeled Cell Number and Morphometric Analysis

Preparation of sections

Sample collection was performed 1 h after ISDN/vehicle injection (Figure 1A). Under anesthesia (25% urethane, 1.5 g/kg i.p.), mice were perfused intracardially, first with saline (0.9% NaCl), then with a paraformaldehyde (PFA) solution (DiaPath Antigenfix P0014, DiaPath, Martinengo, Italy). Brains were then harvested and post-fixed in 4%

PFA for 2 h at 4 °C before being cryoprotected in phosphate-buffered saline 0.1 M (PBS, pH 7.4) containing 30% sucrose and 0.05% sodium azide for at least 2 days at 4 °C. Slices (40 µm thickness) containing the LC region (from -1.54 and -1.40 mm interaural and -5.34 and -5.20 mm relative to the Bregma towards -2.00 and -1.88 mm interaural and -5.80 and -5.68 mm from the Bregma ) (Paxinos and Franklin 2019) were cut using a microtome (MICROM GmbH, Neuss, Germany) and preserved in a solution containing 0.1 M PBS, 40% glycerol, and 0.05% sodium azide at -20 °C until usage.

RESULTS

The TRP-deficient diet reduces TRP but not KYN levels in the plasma

Mice subjected to one week of a TRP-deficient diet showed a significant decrease in TRP concentration compared to physiological levels observed after one week of the BAL diet (Table 2). The percentage of this reduction was similar between groups:

~70.5% for vehicle-injected mice, ~62.0% 1 h after the first ISDN injection (ISDN-1), and ~56.9% 1 h after the third ISDN injection (ISDN-3). Plasma levels of KYN, which is the main endogenous metabolic pathway of TRP (Badawy 2017), were not altered after one week of a TRP-deficient diet consumption in either group compared to vehicle-injected mice subjected to the BAL diet (Table 2). However, the KYN/TRP ratio was augmented in all TRP -groups.

In the LC, mice subjected to one week of the TRP deficient diet also showed reduced TRP concentration (Table 2), with a similar reduction percentage in the three groups:

~79.1% for vehicle-injected mice, ~62.0% 1 h after the ISDN-1, and ~48.1% 1 h after ISDN-3 administrations. KYN levels were also globally reduced as shown by the significant 2-way ANOVA (Table 1), although no individual post-hoc test differences were detected (Table 2). The KYN/TRP ratio was not augmented in all TRP -groups Metabolic reduction after a TRP-deficient diet did not reduce the body weight in any experimental group (Figure S2). Moreover, it did not produce any anxiety-like behavior in the OFT; no differences were found between TRP -and BAL conditions, in the total distance travelled (Figure S3A) or the total time spent in the periphery (Figure S3B).

Moreover, repeated ISDN administrations did not evoke an anxiety-like behavior when compared to vehicle-injected mice.

Repeated ISDN induces persistent CMH which was prolonged by TRP deficiency

The effect of ISDN and/or a TRP-deficient diet on cephalic mechanical sensitivity was measured using the von Frey test (Figure 1). The baseline threshold in mice subjected to the BAL diet was established at 0.93 ± 0.50 g (Figure 1B Left Panel). One hour after ISDN-1, it significantly decreased (0.15 ± 0.02 g), with partial recovery at 3 h (0.47 ± 0.04 g). The baseline threshold before the third ISDN injection was still lower (0.33 ± 0.08 g) than the baseline before ISDN-1, indicating persistent interictal CMH. ISDN-3 also reduced the cephalic threshold that peaked at 1 h post-injection (0.04 ± 0.00 g;

Figure 1B Left Panel) and was similar to the ictal threshold after ISDN-1. The cephalic mechanical threshold returned to baseline level 18 days later (0.32 ± 0.05 g; Figure 1C).

Mice subjected to one week of TRP deficiency showed a baseline threshold (0.87 ± 0.08 g; Figure 1B Right Panel) similar to BAL groups, which was reduced 1 h after the first ISDN injection (0.07 ± 0.02 g). This time, no partial recovery was observed at 3 h (0.08 ± 0.03 g). CMH was also interictally observed before the third ISDN-3 injection (0.16 ± 0.07 g). Repeated ISDN-3 administration kept the threshold lowered (Figure 1B Right Panel), which needed 30 days to achieve total recovery (Figure 1C).

The effect of both diets on ISDN-induced CMH was compared by calculating the AUC of cephalic mechanical thresholds between 1 h and 3 h after ISDN administration (ictal period; Figure 1D1), and between 2 and 30 days after the third ISDN injection (interictal period; Figure 1D2). Indeed, the AUC of both ISDN-1 and ISDN-3 groups submitted to the TRP deficient diet (0.11 ±0.03 AU and 0.10 ± 0.03 AU respectively)

were significantly lower than those from the BAL diet (0.55 ± 0.06 AU and 0.36 ± 0.06 AU) during the ictal period (Figure 1D1). The reduced AUC was also obtained from ISDN-3-injected mice subjected to a TRP-deficient diet for 30 days (16.07 ± 2.29 AU) compared to those following the BAL diet for the same time (6.40 ± 0.98 AU) (Figure 1D2). Moreover, when the time needed to obtain 50% cephalic threshold recovery was estimated for both groups, a marked difference was observed; it took twice as long for mice suffering from a TRP deficiency to reach the same level of recovery (Figure 1E).

Hence, TRP deficiency augments ictal and interictal migraine-like pain induced by single as well as repeated ISDN injections.

The TMF-induced AhR inhibition partially blocks the ISDN-induced CMH

In order to investigate whether the effect of TRP deficiency on cephalic mechanical sensitivity was mediated by AhR, TMF, a potent AhR antagonist was administered 10 min after each repeated ISDN injection. As TMF was injected intraperitoneally, we first validated that it crossed the blood-brain barrier. Therefore, we measured the mRNA level of Cyp1A1, a target gene transcribed after AhR activation. It was significantly reduced after i.p. TMF administration in naïve mice (data not shown), as described before (Cuartero et al. 2014).

TMF alone did not induce pain in vehicle-injected mice subjected to the BAL diet, who showed a similar cephalic threshold throughout the 3 h testing period (Figure 1F1).

TMF injected after the ISDN-1 administration under a BAL regimen has a potential antalgic effect whereby the cephalic sensitivity threshold showed a plateau at ~0.6 g between 1 h and 3 h post-ISDN (Figure 1F1), and the AUC was significantly higher (1.12 ± 0.08 AU) than the group receiving ISDN-1+vehicle (0.44 ± 0.14 AU; Figure 1F1). A similar effect was also shown after repetitive ISDN-3 administrations, although in this case the analgesic effect of TMF was less marked and the cephalic threshold remained ~0.4 g (Figure 1G1), without a significant difference between the AUC of each ISDN-3 group (TMF: 0.73 ± 0.22 AU vs. vehicle: 0.37 ± 0.12 AU; Figure 1F2).

ISDN-1-injected mice subjected to a TRP-deficient diet and receiving TMF showed a reduced cephalic threshold from 1 h to 3 h post-ISDN, although this reduction was less important than that observed in vehicle-injected ISDN mice and ISDN-1+TMF under a BAL diet (Figure S4A). Comparison of the AUC from the groups revealed a significant difference (TMF: 0.55 ± 0.15 AU vs. vehicle: 0.11 ± 0.03 AU; Figure S4B), indicating that TMF did not maintain its analgesic effect under TRP deficiency.

The ITE-induced AhR activation potentiates the ISDN-induced CMH

The systemic administration of ITE, a selective nontoxic AhR agonist that crosses the BBB (Neelamegam et al. 2021) on cephalic mechanical sensitivity was evaluated in mice subjected to BAL or TRP deficient diets. The number of genes differentially regulated by ITE decreases over time, with maximal transcription 6 h post-AhR activation [START_REF] Yan | Aryl Hydrocarbon Receptor Signaling Prevents Activation of Hepatic Stellate Cells and Liver Fibrogenesis in Mice[END_REF]. Hence, we administered ITE 5 h before the first ISDN injection. Under BAL diet conditions, ITE on its own showed no sign of inducing nociception in vehicle-injected mice (Figure 1G1). However, when ITE was followed by a single ISDN administration, the cephalic sensitivity threshold was lower than the threshold from vehicle-injected ISDN mice (Figure 1G1), as confirmed by the reduced AUC (ITE: 0.12± 0.07 AU vs. vehicle: 0.70± 0.12 AU; Figure 1G2). Hence, results

indicate that ITE potentiates the nociceptive action of ISDN. No effect was shown when ITE was coupled with the TRP deficient diet which was further verified by the nonsignificant AUC (data not shown). This could be due to the fact that mice had an initial threshold close to the detection the limit of von Frey technique.

ISDN induces transitory extracephalic mechanical hypersensitivity, which was not altered by TRP deficiency

The effect of ISDN on the mechanical sensitivity of extracephalic regions (left and right Hence, a TRP deficiency doesn't influence the ISDN capacity to induce transitory extracephalic mechanical hypersensitivity.

Both repeated ISDN administration and TRP deficiency provoke functional noradrenergic impairment in the LC

Under pathological conditions, impaired descending inhibition from the LC could contribute to ISDN-induced CMH. Thus, the possible changes after ISDN related to the noradrenergic system were examined by immunohistochemistry, western blot, and HPLC at 1 h after drug administration and one week after the diet started (Figure 1A).

Under a BAL diet, staining with an antibody against TH revealed an increase in the THpositive area (that includes cell bodies and their noradrenergic projections) in the total LC after the third ISDN injection (24.46 ± 2.00 x10 4 µm 2 ) but not after ISDN-1 (20.1 ± 1.60 x10 4 µm 2 ) (Figures 3A1 and3A2). When the LC was divided into rostral, middle, and caudal sections, no differences were observed between groups (Table S2). The increased TH surface in ISDN-3-injected females was not correlated neither with an increase in the number of TH-IR cells, which was similar between all BAL-diet groups (Figures 3A3), nor with an increase of TH expression measured by western blot (Figure 3B). NA levels measured by HPLC were also similar between ISDN and vehicleinjected mice (Figure 3C).

One week after the TRP-deficient diet did not induce any change in the number of detected TH-IR cells (Figure 3A3) nor in their projection area (Figure 3A2). However, TRP deficiency increased TH expression in both vehicle and ISDN-injected groups (vehicle: 8.4 ± 1.0 x10 7 , ISDN-1: 8.0 ± 1.1 x10 7 , ISDN-3: 8.3 ± 1.3 x10 7 ; Figure 3B) compared to the BAL-diet groups (vehicle: 5.6 ± 0.7 x10 7 , ISDN-1: 5.4 ± 0.8 x10 7 , ISDN-3: 4.6 ± 1.1 x10 7 ; Figure 3B). TRP deficiency also altered the production of NA, which was significantly lower in ISDN-1 (141.7 ± 8.8 nM) and nM) than in vehicle-injected mice (186.7 ± 17.4 nM; Figure 3C)

Both repeated ISDN and TRP deficiency provoke morphological changes in noradrenergic LC neurons

It has been described that the LC neurons express AhR (E. Kimura et al. 2021).

Therefore, the effect of ISDN alone or coupled with a TRP-deficient diet on AhR function in noradrenergic LC neurons was evaluated. First, LC cells that expressed AhR were identified by immunohistochemistry (Figure 4A). Under a BAL diet, 50.4 ± 1.9 % of TH-IR cells co-expressed AhR in vehicle-injected mice (Figure 4B). A similar cell proportion was observed in the three LC sections (Table S3). A total of 253 AhR + TH-IR neurons (pooled from 5 vehicle-injected mice under a BAL diet) were characterized by a cell body area of 160.5 ± 3.4 µm 2 (Figure 4C1) with a nuclear area of 47.9 ± 1.1 µm 2 (Figure 4C2).

Second, the effect of ISDN and/or TRP deficiency on TH-IR cells was analyzed. Under a BAL-diet, ISDN-3 increased the global size of AhR + TH-IR, both the cell body (189.3 ± 6.3 µm 2 ; Figure 4C1) and nuclear areas (57.8 ±2.1 µm 2 ; Figure 4C2), compared to the vehicle-injected mice (160.5 ±3.4 µm 2 and 47.9 ± 1.1 µm 2 respectively). This change was predominately at the level of the middle and caudal LC (Table S3). No changes were observed after ISDN-1.

AhR expression and activation are altered after ISDN administration in the LC

AhR expression in the total LC, including TH-IR neurons and glial cells that also expressed AhR, was quantified by RT-PCR. Reduced mRNA expression was observed in ISDN-3 groups (BAL diet: 69.8 ± 13.0 and TRP deficient diet: 65.9 ± 17.5, as shown by the significant 2-way ANOVA (Table 1), although Tukey post-hoc test did not reveal significant intergroup differences (Figure 4D). TRP deficiency did not modify AhR expression in vehicle-injected mice and does not have a cumulative effect in mice receiving an ISDN administration.

The activation of AhR induces its translocation to the nucleus, where it activates the transcription of target genes (Hubbard et al. 2015). Thus, the expression of AhR immunolabelling in the soma and the nucleus of AhR + TH-IR cells was quantified. The ratio (in %) of labeling in the nucleus to that of the cytoplasm was calculated following the method described by (E. Kimura et al. 2021) and used as an indirect measure of AhR activation. A decrease in the percentage of nuclear intensity of AhR was observed in ISDN-3-injected mice subjected to the BAL diet (31.4 ± 0.6 %) compared to the vehicle group (32.3 ± 0.78 %; Figure 4E). This reduction was mainly detected at the caudal level of the LC (Table S3). No differences were observed in mice subjected to a TRP-deficient diet.

The AhR activity is blocked by the AhR repressor (AhRR), which is transcribed after AhR activation to induce a negative control (Mimura et al. 1999). Indeed, a significant effect of ISDN on AhRR expression was detected by 2-way ANOVA (Table 1), as

shown by increased mRNA levels in the ISDN-3 groups (Figure 4F), although the Tukey post-hoc test did not reveal significant intergroup differences. TRP deficiency did not modify AhRR expression in vehicle-injected mice and also does not have a cumulative effect in the ISDN-injected groups.

Finally, the mRNA expression of two main target genes of AhR, Cyp1A1, and Cyp1B1, were also examined by RT-PCR (Figure 4G Left Panel). Cyp1A1 expression was unaltered in any of the conditions showing similar levels in all groups (vehicle and ISDN), in both BAL and TRP deficient conditions. However, Cyp1B1 showed a significant ISDN effect by 2-way ANOVA (Table 1), which reduced mRNA levels in ISDN-3 groups without Tukey post-hoc test intergroup differences (Figure 4G Right Panel). Vehicle-injected mice under a TRP-deficient diet showed comparable Cyp1B1 expression to that of the BAL group, indicating that a TRP-deficient diet does not induce changes in the expression of Cyp1B1.

Repeated ISDN coupled with TRP deficiency increases microglial reactivity in the LC

AhR activity is linked to various immunological responses, which can contribute to central sensitization and cephalic and extracephalic hypersensitivities (Ji et al. 2018). At the level of glial cells, analysis of the morphology and quantification of cell number are commonly used as single measures to report on the activation state of microglia and astrocytes. Thus, we examined the possible glial changes at 1 h after ISDN administration and/or one week of TRP deficiency by immunohistochemistry (staining with an antibody against IBA1 for microglia and against GFAP for astrocytes) at the level of the LC.

Analysis of IBA1 labeling was separately done in the area occupied by noradrenergic cell bodies and in the TH-IR branching area. As no differences were observed between both LC areas, data were added together. Under a BAL diet, no change was seen in the number of quantified IBA1-IR cells in the total TH-IR region (Figure 5A and 5B), neither in any of the three portions nor after ISDN-1 or ISDN-3 groups. TRP deficiency also did not modify the microglia density. However, concerning the average size of the microglia, the sole change could be seen with a significant increase in the body cell size 1 h after the third ISDN injection in mice subjected to a TRP deficient diet (109.20 ± 2.30 µm 2 ) compared to its respective vehicle-injected mice (90.10 ± 2.00 µm 2 ) (Figure 5C). Morphological changes were also found at the level of the middle (vehicle: 88.00 ± 1.87 µm 2 vs. ISDN-3: 110.27 ± 3.00 µm 2 ) and caudal (Vehicle: 91.46 ± 2.80 vs. ISDN-3: 106.87 ± 3.21) portions of the LC (Table S6). Hence, signs of microglial activation occur under a combined effect of both TRP deficiency and repetitive ISDN administrations.

Repetitive ISDN administrations induce astrogliosis in the LC

Like microglia, analyses of GFAP labeling included the area occupied by noradrenergic cell bodies and the TH-IR branch area. A significant increase in the GFAP immunoreactivity was detected after both the first (4.56 ± 0.37) and the third ISDN injections (3.75 ± 0.29) compared to vehicle-injected mice under a BAL diet (2.15 ± 0.28; Figure 6A and6B). The same increase in GFAP-IR was found after ISDN-1 (4.66 ± 0.21) and ISDN-3 (4.17 ± 0.34) with respect to the vehicle (2.59 ± 0.42) under the TRP deficient diet (Figure 6B). No changes were found in the reactivity of GFAP under TRP-deficient conditions compared to their BAL food counterparts. Looking at the three portions of the LC, the same reactivity seen in the total LC can be seen primarily at the level of the rostral and middle portions (Table S5).

The density of the astrocytes showed no changes neither with ISDN nor with a TRPdeficient regimen in the total LC (Figure 6C) and as well in every portion of it (Table S5). However, the cell body size of the astrocytes showed a significant change after three ISDN injections in mice subjected to a BAL regimen that could be seen at the level of the total LC (Veh: 29.70 ± 1.35; ISDN: 40.19 ± 0.90) (Figure 5D) and also at the level of all rostrocaudal subregions (Table S5). Hence, ISDN provokes astrocytic reactivity under a BAL food regimen, with the TRP deficiency showing no specific effect on this reactivity. Moreover, the effect of ISDN is interestingly limited to the rostral and middle portions of the LC, with a slight change at the level of the caudal LC.

After a noxious stimulation, astrocytes undergo a series of phenotypic and functional changes such as process extensions and hypertrophy, which form the phenotypic characteristics of reactive astrocytes (T. Li et al. 2019). Our results showed increased cell body size of astrocytes after ISDN-3. Hence, we reconstructed a total of 166 GFAP-IR astrocytes to analyze their 3D morphology (Figure 7A). The morphometric assessment gave rise to the following four parameters for the total LC and the three rostrocaudal regions from each group: the fractal dimension, the number of total branches, the average branch length, and the area covered for each astrocyte. Vehicleinjected mice subjected to the BAL diet showed astrocytes with a fractal dimension of 1.11 ± 0.01 (Figure 7B), a number of branches of 18.8 ± 1.0 (Figure 7C), and an average length of 11.1 ± 0.7 µm (Figure 7D). The maximal number of intersections (peak of the Sholl plot) was at an average distance of 10 μm to the cell body (Figure 7F1 Left Panel). One ISDN injection induced some morphological changes, including a reduction of the branch number (14.3 ± 1.0; Figure 7C) and therefore, of their area (0.6 ± 0.1 µm 2 ; Figure 7E), and a remodeling of cell process distribution: the number of intersections was reduced, in particular at distances between 10 to 30 μm around the body cell. The peak of the Sholl plot reduced in amplitude and shifted to small distances (~5 µm; Figure 7F1 Left Panel). After the third ISDN injection, astrocytes were again remodeled: they presented reduced cell complexity, as shown by the reduced fractal dimension (1.05 ± 0.01; Figure 7B) compared to vehicle-injected mice (1.11 ± 0.01).

Changes in the morphological organization were associated with a reduced number of branches (14.20 ± 0.80; Figure 7C), which were longer (13.10 ± 0.50 µm; Figure 7D).

Hence, the area covered by each astrocyte also increased (Figure 7E). Moreover, they showed fewer interactions of vehicle-injected mice (Figure 7F2), with a Sholl peak around 10 µm of distance (Figure 7F1 Left Panel).

TRP deficiency reduced the number of cell processes (14.90 ± 0.80; Figure 7C) in vehicle-injected mice, with no significant change in the number of intersections (Figure 7F1 Right Panel) compared to vehicle-injected mice under a BAL diet (Figure 7F2).

TRP deficiency also did not potentiate the morphological remodeling induced by ISDN-1 or ISDN-3, as shown by similar morphometric values as those of astrocytes from ISDN groups under the BAL diet. Together, these data indicate that ISDN alone or coupled with TRP deficiency induces rapid astrocytic activation in the LC, which was observed in the three rostrocaudal subregions (Table S6).

Both ISDN administration and TRP deficiency increase plasma levels of proinflammatory cytokines

Reactive microglia and astrocytes are associated with the release of pro-inflammatory cytokines, whose levels have been described to be increased in both episodic and chronic migraineurs (Aydın et al. 2015). Therefore, the plasma concentrations of four pro-inflammatory cytokines were measured: IL-1β and IL-6, TNFα which are mainly released by activated astrocytes and microglia (Liddelow and Barres 2017), and IFNγ, which is released by activated microglia (Lehnardt 2010). Following one ISDN administration, a significant increase was shown in the levels of IL-1β (67.28 ± 2.84 pg/mL), TNFα (447.5 ± 11.7), and IL-6 (95.4 ± 3.54 pg/mL) compared to vehicleinjected mice (26.75 ± 2.09 pg/mL, 257.82 ± 15.21 pg/mL, 46.76 ± 3.91 pg/mL respectively; Table 3). The same increase in the plasma concentration was significantly shown after repetitive ISDN administrations with respect to IL-1β (76.55 ± 6.24 pg/mL), TNFα (439.45 ± 11.24 pg/mL), and IL-6 (89.98 ± 3.08 pg/mL), with an effect not more robust than that of one ISDN administration (Table 3). Interestingly, TRP deficiency induced the same inflammatory increase of all three cytokines IL-1β in vehicle-injected mice (Table 3). However, it did not augment the effect of ISDN as mice showed comparable cytokine levels under a TRP-deficient diet compared to the BAL diet (Table 3). No changes were seen with respect to IFNγ either after ISDN or under all the remaining conditions (Table 3). Hence, based on the prominent increase in the levels of pro-inflammatory cytokines, both ISDN and TRP deficiency induce a state of general inflammation.

DISCUSSION

In this study, we demonstrated that ISDN induces a pro-inflammatory state shown by the systemic pro-inflammatory cytokine release, enhanced astrogliosis, and altered AhR function in the LC. TRP deficiency also increased plasmatic pro-inflammatory cytokines. Combined, ISDN and TRP deficiency worsen the pro-inflammatory state and impaired the noradrenergic function of the LC, which was concomitant with a longlasting CMH. Moreover, we highlight that the inflammatory-related pain facilitation during migraine progression was linked to the pro-nociceptive AhR activity.

CMH is concomitant with an abnormal systemic and local inflammatory state

Systemic administration of ISDN induced a robust acute CMH and EMH in females, in accordance with our published work showing that ISDN elicits both cephalic and extracephalic hypersensitivities in rats (Dallel, Descheemaeker, and Luccarini 2018). In our results, ISDN-induced CMH lasted for approximately 14 days while EMH was reversed after 4 days. This has priorly been shown in an inflammatory soup model of migraine-like pain whereby repetitive stimulations induce a persistent CMH but a reversible EMH probably due to their mechanistic differences in reference to sensitization (Boyer et al. 2014). This distinction is due to the different mediators of sensitization being central sensitization of the Sp5c as a mediator of cephalic cutaneous hypersensitivity while the thalamic trigeminovascular neurons are responsible for extracephalic cutaneous hypersensitivity.

Enhanced inflammatory activity under ISDN conditions was shown by significant astrogliosis in the LC coupled with increased levels of pro-inflammatory cytokines in the plasma. Several features have been described to be associated with astrogliosis, such as the up-regulation of GFAP and cellular hypertrophy (Sofroniew 2015). Our analyses showed that single and repeated ISDN administrations increased GFAP cell reactivity, as described before after nitroglycerine (NTG) administration in mice (Filippone et al. 2022). Repeated ISDN also altered the morphology and reduced the arbor complexity of the astrocytes in the LC, matching with the peak of CMH. Morphological changes in astrocytes haven't been shown under migraine-like conditions, however, in conditions of harmful insults astrocytes become more reactive and change their shape through hypertrophying and processes extension towards the area of insult to perform their role of protection under chronic pain conditions (D. Sun and Jakobs 2012, 20;Y. Wang et al. 2018). Although microglial activation has been described to be related to astrogliosis (D. Zhang et al. 2010), our results did not reveal increased microglial reactivity after ISDN (as far as size or cell number are concerned). However, changes in gene expression or the release of inflammatory molecules by microglia without morphological alterations cannot be excluded. Indeed, after both single and repeated ISDN administrations, increased plasmatic levels of certain pro-inflammatory mediators including IL-6, TNFα, and IL-1β were measured, which are mainly released by activated astrocytes but also microglial cells (Liddelow and Barres 2017). NO has been also described to stimulate cytokine release (Soufli et al. 2016). Since both astrogliosis and cytokine levels were measured at the same time point after ISDN administration, it is difficult to determine the temporal sequence of events, and further studies will be necessary in order to analyze if systemic cytokine release came from glial activation, ISDN-derived NO, or from a combination of both.

Dysregulated TRP metabolism promotes systemic inflammation and worsens

CMH

Acute dietary TRP depletion has been used as a tool for clinical and preclinical research about the role of TRP-derived metabolites in migraine pathophysiology. In humans, TRP depletion augmented clinical manifestations of migraine, such as nausea, headache intensity, and photophobia (P. Drummond 2006). In rodents, TRP depletion has previously and reproducibly induced neurochemical and behavioral alterations in both rats (Cahir et al. 2007) and mice (Browne et al. 2012). TRP is the precursor of serotonin and KYN, which have a prominent role in migraine (Berman et al. 2006;K. Gao et al. 2020). These metabolites play a role in the pathophysiology of migraine and are altered in migraineurs. To mimic this clinically persistent TRP dysregulation in mice, we used a chronic dietary TRP depletion, which reduced approximately 70% of the plasmatic TRP concentration after one week, without affecting anxiety or the welfare state of animals.

It has been described that TRP metabolism modulates peripheral inflammatory responses, at the level of the gut-brain axis (Bosi et al. 2020), through AhR activation.

We observed that TRP deficiency increased IL-6, TNFα, and IL-1β levels in the blood, as described before (van Wissen et al. 2002), maybe by activating AhR. Proinflammatory cytokines, mainly TNFα (Gostner et al. 2020), activate the enzyme indoleamine 2,3-dioxygenase (IDO) in peripheral dendritic cells, which transforms TRP to KYN. IDO plays a minor role under physiological circumstances. However, it is strongly activated upon inflammation (Yeung et al. 2015). IDO activity is often measured by the blood KYN/TRP ratio, which is increased in diseases characterized by excessive or chronic inflammation (Schröcksnadel et al. 2006). Our data showed that ISDN also increased peripheral IDO activity thus shifting TRP metabolism toward the production of KYN, leading to low TRP levels, and increased KYN/TRP ratio. This effect could be explained by an indirect action of ISDN on IDO activity: NO produced from ISDN could stimulate TNFα release, which in turn, activates IDO that metabolizes TRP to KYN (Figure 8A). Impaired turnover of KYN and TRP has been described in migraineurs, who presented low levels of both metabolites compared to healthy controls (Tuka et al. 2021).

Both levels of TRP and KYN were reduced after ISDN and/or TRP-depleted diet in the LC. IDO expression in the brain is low and restricted to specific brain regions (Kwidzinski and Bechmann 2007). Cerebral TRP metabolism is therefore largely dependent on the transport of TRP and KYN across the blood-brain barrier. Both have access to brain tissue through the large neutral amino acid transporter, hence entering through a competition with other large neutral amino acids leading to their marginally lower levels.

KYN is a ligand of AhR and regulates inflammatory processes through the promotion of cytokine release and hence through the further activation of the IDO creating a cyclic event (Figure 8A). This process included the activation of astrocytes and microglia, which progress to a reactive morphological state when both TRP deficiency and repetition of ISDN were combined. NO also controls the activity and expression of AhR by decreasing it (Niedbala et al. 2011). Thus, the final balance between KYN and NO actions on AhR could be a partial reduction of AhR expression after ISDN, leaving enough active AhR to promote the pronociceptive effect observed after its direct activation by ITE. This enhanced pro-inflammatory state could be a potential inducer of pain facilitation and persistence, as observed by long-lasting CMH in mice after TRP deficiency. It should be noted that TRP deficiency did not affect ISDN-induced extracephalic hypersensitivity since the targeted pathway is different. This difference could be due to the different mechanisms of both hypersensitivities considering in episodic migraine cephalic hypersensitivity is mostly mechanical, whereas extracephalic is mostly thermal (Guy et al. 2010).

AhRs have a pro-nociceptive action in migraine pathophysiology

AhRs are prominent transcription factors of either pro-or anti-inflammatory capacities, that are located at the level of the astrocytes, microglia, and noradrenergic neurons. In most inflammatory diseases, AhR has been described as neuroprotective by regulating the neuroinflammatory responses in the brain (Ntranos et al. 2022). For instance in an animal model of chronic constrictive nerve injury the deletion of AhR worsened the damage, while the administration of an AhR agonist counteracted such changes (Sheu et al. 2022).

Interestingly, our data showed that AhR has a pro-nociceptive action during migraine initiation and progression. On the one hand, systemic activation of AhR by the nontoxic agonist ITE, prior to ISDN administration, enhanced the intensity and duration of ictal CMH. On the other hand, AhR inhibition by non-toxic TMF prevents both ictal and interictal ISDN-induced CMH. Our hypothesis is that AhR activation promotes the sensitization of the inflammatory signaling pathways, which respond inordinately after ISDN administration, facilitating nociceptive response and central sensitization of the trigeminal system (Figure 8B). Since AhR is blocked by TMF, KYN can no longer execute its function as a ligand, and hence the IDO/AhR axis is potentially hindered (Figure 8C). Supporting this idea, the combination of TMF with a TRP-deficient diet (that prior activates AhR and hence sensitizes the peripheral inflammatory system) leads to a lower TMF-induced antalgic effect.

Impaired noradrenergic function contributes to migraine progression

Multiple players have an important role in the pathophysiology of migraine including the LC. This nucleus plays an important role in the descending control of pain during migraine. Moreover, its role in migraine has been shown in neuroimaging studies

showcasing altered LC functional connectivity in migraineurs (Moulton et al. 2014), and LC activation following the infusion of NTG infusion (Maniyar et al. 2014). In our study, we observed that the noradrenergic function of the LC neurons was altered under TRP deficiency. Indeed, TH expression was increased in ISDN-injected mice coupled with reduced local NA release. TRP depletion has been described to reduce NA levels in rodents (Ardis et al. 2009), although the exact mechanism is not clear. It could involve altered serotoninergic modulation of LC activity (Deen et al. 2018) or enhanced turnover of NA, as described in other chronic pain models (Alba-Delgado et al. 2016).

In any case, NA has an endogenous analgesic function in the spinal cord (Pertovaara 2006), and low release could contribute to long-lasting CMH induced by TRP deficiency in ISDN-injected mice.

The effect of TRP metabolism disruption also alters the noradrenergic cell morphology.

Indeed, after repeated ISDN administrations, an increase in the cell volume of noradrenergic neurons and more TH-IR projections were observed. However, TRP deficiency blocked these structural alterations, leading to a concomitant long-lasting CMH. These results suggest that morphological changes of LC neurons are necessary to modulate nociceptive responses. Indeed, several studies have described morphological changes in LC neurons in response to stressful stimuli (Borodovitsyna, Joshi, and Chandler 2018). These alterations include an increase in the complexity of the dendritic arborization associated with the expression of genes involved in cell structure and synaptic plasticity with the primary aim of pain amelioration and modulation.

CONCLUSION

This study demonstrates that migraine-associated CMH is linked to an enhanced neuroinflammatory state promoted by impaired TRP metabolism and comorbid with noradrenergic dysfunction. Moreover, we propose AhR as a pivotal player in TRPmediated inflammatory signaling with a potential pro-nociceptive role. Other studies will be necessary to dive into the underlying circuit of AhR activation during migraine initiation and progression, and thus propose future therapeutic targets. ) or not 2 or 3-way ANOVA with corresponding degrees of freedom (DFn, DFd), and p-values. ANOVA factors were designed as followed: Time, for comparisons between different time points; Treatment, for comparisons between TMF or ITE; Diet, for comparisons between BAL and TRP -; ISDN, for comparisons between ISDN and Vehicle; NB-INJ, number of injections, for comparisons between animals receiving one or three injections; Distance, for the distance from the cell body, Interaction for effect between two or three factors on the dependent variable. p <0.05 was considered statistically significant. R: Right; L: Left.

COMPLEMENTARY RESULTS

In this section, additional experiments that were not included in the two primary studies are presented. or TRP-deficient (TRP -) diets.

I. Study of

Glial quantification was performed according to the total structure or divided into the three subdivisions of the Sp5c: V1 (innervated by the ophthalmic branch of the trigeminal nerve), V2 (innervated by the maxillary branch), and V3 (innervated by the mandibular branch).

I.1. Astrocytic Activity

ISDN administration did not induce changes in the GFAP intensity in any of the three regions of the Sp5C (Table 1). However, TRP deficiency globally reduced the GFAP labeling in the vehicle and ISDN-injected mice (Table 1). Changes were observed mainly in V1 and V3 regions.

I.2. Microglial Activity

Assessment of the microglial activity showed no change after the administration of ISDN in the microglial density in any of the three regions of the Sp5C (Table 2).

Following one week of a TRP-deficient diet, no sign of a change was also recorded.

I.3. AhR Activity

Assessment of the relative levels of AhR mRNA expression and its respective target genes was done under ISDN-1 and ISDN-3 administrations and a BAL or TRPdeficient diet. The results are presented in the following Table 3, where no changes were observed.

II. Study of the effect of TRP supplementation on ISDN-induced cephalic mechanical hypersensitivity

Based on the data obtained throughout this project, we show that the deficiency of TRP is a contributing factor to the augmentation of the cephalic mechanical hypersensitivity (CMH) induced by ISDN. However, the question arises about what would occur if we introduce again TRP in the diet (at the standard dietary level) after ISDN-induced CMH in female mice. Hence, we applied the following protocol: after one week of a TRP-deficient diet, mice were administered with three repetitive ISDN administrations over two days. 72 hours post ISDN-3, the TRP-deficient diet was changed by a BAL diet containing 2.3 g of TRP (Figure 1A). Measurements of the cephalic mechanical sensitivity were done over 30 days to assess the duration of pain persistence. Moreover, we assessed the levels of TRP in the serum 24 h following the TRP supplementation.

Figure 1B showcases the established result in Study 2 that following a TRP deficiency, ISDN-3-injected animals needed 30 days to achieve total recovery of the cephalic mechanical threshold. Re-supplementing the system with TRP, interestingly showed a vast recovery at an approximate time similar to that of mice under a BAL diet (Figure 1B). This was confirmed by the similar AUC of TRP Supp mice that is higher than that of the TRP-deficient counterpart (Figure 1C). Concerning the serum TRP concentration, a significant increase was shown 24 h after the switch from TRPdeficient to the BAL diet, showing similar plasma TRP levels to that of the ISDNinjected mice subjected to the BAL diet and higher than that of the TRP-deficient diet (Figure 1D). Within this thesis project, we worked on two central hypotheses, starting with that cephalic mechanical hypersensitivity is coupled with an enhanced inflammatory response that contributes to migraine progression to a chronic state and affects the abortive efficacy of antimigraine drugs. Then we moved toward our second one whereby we hypothesize that TRP-deficient metabolism coupled with an impaired AhR activity could play a role in neuroinflammation during migraine progression. Our work was divided between two primary studies whereby we combined behavioral assessment (pain, light aversion, and anxiety), immunohistochemistry, and biomolecular techniques (RT-PCR, HPLC, western blot, and ELISA) and focused on the level of the LC and Sp5c in an experimental mouse model of migraine-like pain induced by systemic ISDN administrations.

Throughout this journey, we were able to show that:

(1) A single systemic ISDN administration evokes a transitory CMH in both male and female mice, and LH only in females. Both migraine-related symptoms were sensitive to sumatriptan treatment.

(2) Three repeated systemic ISDN administrations induce persistent CMH in female mice, which was concomitant with a systemic increase of plasmatic pro-inflammatory cytokine levels and a local increase of astrocytic reactivity in the LC. (4) TRP-mediated long-lasting ISDN-induced CMH was concomitant with functional impairment of the serotoninergic system in the Sp5C (increased levels of 5-HTR1D after acute ISDN administration), and the noradrenergic activity in the LC (reduced NA release after ISDN-3), and with both astrocytic and microglial enhanced activation.

(5) Finally, AhR plays a pro-nociceptive role in the initiation and progression of ISDN-induced CMH.

ISDN as a Preclinical Model of Migraine

Several preclinical models of migraine-like pain have been developed to study the pathophysiological mechanisms of migraine. We privileged the ISDN model for several reasons. First, ISDN causes headaches in migraineurs but not in healthy subjects (Bellantonio et al. 1997;Castellano et al. 1998), compared to NTG which is less specific [START_REF] Ramachandran | A Naturalistic Glyceryl Trinitrate Infusion Migraine Model in the Rat[END_REF]. Second, we previously validated ISDN as a predictive preclinical model of migraine in male rats (Dallel, Descheemaeker, and Luccarini 2018;Flores Ramos et al. 2017). Third, ISDN-induced CMH was correlated with central sensitization in the Sp5C in rats (Flores,2017). In our study, we tested for the first time ISDN in male and female mice. We discovered that, similar to rats, a single ISDN injection elicited reversible CMH and EMH, while repeated ISDN administrations induce persistent CMH. Persistent CMH supports the clinical profile where when the frequency of headaches is more recurrent the higher the chance is to progress from an acute to a chronic state (Ann I. [START_REF] Scher | Risk Factors for Headache Chronification[END_REF].

EMH was reversible with no sign of persistence which has been priorly shown in an inflammatory soup model that demonstrated that both hypersensitivities are of a different mechanistic nature (Boyer et al. 2014). These mechanistic differences could be attributed to sensitization (Guy et al. 2010). Furthermore, ISDN only elicited LH in females. This light aversive behavior is similar to that following an acute NTG injection which has previously been described in both male (Markovics et al. 2012) and female mice (Eller et al. 2021). The lack of LH in males could be attributed to the fact that females are more sensitive to NO donors than males, as shown by the higher activity of the NO synthase in females compared to males (Oydanich et al. 2019). Moreover, males showed a higher innate sensibility to light than females, which could support different behavioral and neuronal mechanisms for light adaptation (Chellappa 2021).

ISDN Induces an Abnormal Neuroinflammatory State

Emerging data also suggest that neuroinflammation is a modulator of central sensitization. Increased levels of pro-inflammatory chemokines and cytokines have been detected during ictal and interictal periods in migraine sufferers (Biscetti et al. 2022). In rats, NTG-induced CM was correlated with increased transcriptional activity of IL-6 and TNFα in the trigeminal ganglia and pons (including the LC) (Greco et al. 2017), and with microglial activation in the Sp5C, contributing to central sensitization (He et al. 2019). In our model, a single ISDN administration did not evoke inflammatory responses, while repeated ISDN injections induce an inflammatory reactivity, exhibited by a substantial increase in GFAP reactivity, astrocytic morphological alterations in the LC, and increased levels of pro-inflammatory cytokines at the plasma levels. When compared to sham controls, considerable amounts of cytokine release were observed post-NTG induction in several migraine models, including the robust NTG model (Filippone et al. 2022). Furthermore, we see a considerable morphological change in the astrocytes, with a decrease in complexity and an increase in area and length. Morphological alterations in astrocytes have not been seen in migraine patients. In the presence of damaging insults, however, astrocytes become more reactive and modify their morphology by hypertrophying and process extension towards the location of damage to perform their duty of protection (D. Sun and Jakobs 2012). However, at the level of the Sp5c, no sign of change was seen with respect to both astrocytic and microglial activity. It could be postulated that since the LC has an integral role in the modulation of descending controls of pain, the first step of reactivity is shown at the level of the LC following repeated administrations of the NO donor.

Chronic TRP deficiency is a Contributor to Migraine

Pathophysiology and Neuroinflammation

Cephalic, Extracephalic, and Light Hypersensitivities

Tryptophan deficiency has demonstrated clinical evidence as a pivotal player in migraine pathogenesis, primarily in the enhancement of the symptoms of migraine (nausea, headache severity, and photophobia) (P. Drummond 2006). We dysregulated TRP metabolism before inducing migraine, and found a substantial increase in pain persistence, both in acute and interictal periods. This is consistent with clinical models in which dietary acute TRP depletion increases headache severity (P. Drummond 2006), but no research has established its function in pain persistence. Within this project however, we have been able to postulate that TRP deficiency creates an inflammatory state that is further augmented by the action of the NO provided by the administration of ISDN, which I will explain in a further section within the discussion.

TRP deficiency, on the other hand, did not affect ISDN-induced EMH; since the targeted pathway is of a different nature. This disparity in nature might be related to the distinct processes of both hypersensitivities and different manifestations considering in episodic migraine cephalic hypersensitivity is mostly mechanical, whereas extracephalic is mostly thermal (Guy et al. 2010). However, a TRP-deficient diet did not aggravate the ISDN-induced LH in females. This contradicts what the literature has shown as to an increase in LH in migraineurs post-TRP depletion (P. Drummond 2006). This could be due to the pathway targeted by TRP. Since CMH and LH are two distinct pathological pathways, the effect of a TRP-deficient diet could be solely targeting CMH. Moreover, it could be attributed to the timing of the assessment of light hypersensitivity post-ISDN administration, which was one-hour post-ISDN, perhaps assessment at a different time point would showcase an effect on LH.

Neuroinflammation

The metabolism of TRP is linked to aging and generates metabolites that modulate inflammation, maintain energy homeostasis, and influence behavior [START_REF] Cervenka | Kynurenines: Tryptophan's Metabolites in Exercise, Inflammation, and Mental Health[END_REF]. TRP metabolites also have a role in the regulation of tissue damage and inflammation. Serotonin is one example, which has been linked to intestinal inflammation [START_REF] Spohn | Non-Conventional Features of Peripheral Serotonin Signalling -the Gut and Beyond[END_REF]. Modulations in the levels of TRP, such as its deficiency have been shown to compromise immune responses and impair disease resistance in teleost fish [START_REF] Machado | Dietary Tryptophan Deficiency and Its Supplementation Compromises Inflammatory Mechanisms and Disease Resistance in a Teleost Fish[END_REF]. Furthermore, gut health and intestinal immunity require sufficient dietary TRP for efficient immunological responses and intestinal homeostasis (J. [START_REF] Gao | Impact of the Gut Microbiota on Intestinal Immunity Mediated by Tryptophan Metabolism[END_REF]. TRP metabolites are known to support gut microbiota, microbial metabolism, the host's immune system, and hostmicrobiota synergy [START_REF] Lamas | CARD9 Impacts Colitis by Altering Gut Microbiota Metabolism of Tryptophan into Aryl Hydrocarbon Receptor Ligands[END_REF]).

In our results, changes in microglial reactivity required the concomitant impact of TRP dysregulation and repeated ISDN administrations. Understanding the difference between astrocytic and microglial responses requires some understanding of proinflammatory cytokine levels. ISDN and TRP both elevated pro-inflammatory mediators such as IL-6, TNF, and IL-1, but not IFNγ. TNF, IL-1, and IFNγ are mostly released by activated astrocytes and microglia, respectively (Liddelow and Barres 2017). As a result, the absence of change in IFNγ expression suggests that the action of ISDN and TRP alone is inadequate to induce activation. Furthermore, the astrocytes govern the activity of the microglia, meaning that this burst of activity is contingent on an already active astrocytic population.

Noradrenergic Impairment

The LC is an integral noradrenergic center in migraine pathophysiology. Under TRP deficient conditions, we observed that the noradrenergic function of the LC neurons was altered . The TH expression was increased in ISDN-injected mice coupled with a reduced local NA release. TRP depletion has been described to reduce NA levels in rodents (Ardis et al. 2009), although the exact mechanism is not clear. It could involve altered serotoninergic modulation of LC activity (Deen et al. 2018) or enhanced turnover of NA, as described in other chronic pain models (Alba-Delgado et al. 2016).

NA has an endogenous analgesic function in the spinal cord (Pertovaara 2006), and low release could contribute to long-lasting CMH induced by TRP deficiency in ISDNinjected mice.

Our results also suggest that morphological changes of LC neurons are necessary to modulate nociceptive responses. We have shown that the effect of TRP metabolism disruption also alters the noradrenergic cell morphology. Repeated ISDN administrations, induce an increase in the cell volume of noradrenergic neurons and more TH-IR projections were observed. However, TRP deficiency inhibited these structural alterations, leading to a concomitant long-lasting CMH. A plethora of studies have described morphological changes in LC neurons in response to stressful stimuli (Borodovitsyna, Joshi, and Chandler 2018). These alterations include an increase in the complexity of the dendritic arborization associated with the expression of genes involved in cell structure and synaptic plasticity with the primary aim of pain amelioration and modulation.

Further insight into the role of the LC will be through local NA inhibition and through electrophysiological analysis in mice under migraine-like conditions.

The TRP-AhR Axis

The action of TRP-derived metabolites might be mediated through AhR since they are endogenous ligands. AhR are important transcription factors with either pro-or anti-inflammatory properties that are found in astrocytes, microglia, and noradrenergic neurons. Hence, coupling the reactivity induced by ISDN and the modulatory role of AhRs we wanted to assess whether the changes occurring due to TRP depletion occur as a function of mechanistic changes at the level of LC AhRs. ISDN is a potent NO donor, and we know that NO promotes the systemic release of pro-inflammatory cytokines, including IL-6, IL-1β, and TNFα in the blood (Soufli et al. 2016). We observed that TRP deficiency increased IL-6, TNFα, and IL-1β levels in the blood, as described before (van Wissen et al. 2002), maybe by activating AhR. Pro-inflammatory cytokines, mainly TNFα (Gostner et al. 2020), activate the enzyme indoleamine 2,3-dioxygenase (IDO) in peripheral dendritic cells, which transforms TRP to KYN. IDO plays a minor role under physiological circumstances. However, it is strongly activated upon inflammation (Yeung et al. 2015). IDO activity is often measured by the blood KYN/TRP ratio, which is increased in diseases characterized by excessive or chronic inflammation (Schröcksnadel et al. 2006). Our data showed that ISDN also increased peripheral IDO activity thus shifting TRP metabolism toward the production of KYN, leading to low TRP levels, and increased KYN/TRP ratio. This effect could be explained by an indirect action of ISDN on IDO activity: NO produced from ISDN could stimulate TNFα release, which in turn, activates IDO that metabolizes TRP to KYN. Impaired turnover of KYN and TRP has been described in migraineurs, who presented low levels of both metabolites compared to healthy controls (Tuka et al. 2021).

Both levels of TRP and KYN were reduced after ISDN and/or TRP-depleted diet in the LC. IDO expression in the brain is low and restricted to specific brain regions (Kwidzinski and Bechmann 2007). Cerebral TRP metabolism is therefore largely dependent on the transport of TRP and KYN across the blood-brain barrier. Both have access to brain tissue through the large neutral amino acid transporter, hence entering through a competition with other large neutral amino acids leading to their marginally lower levels.

KYN is a ligand of AhR and regulates inflammatory processes through the promotion of cytokine release and hence through the further activation of the IDO creating a cyclic event. This process included the activation of astrocytes and microglia, which progress to a reactive morphological state when both TRP deficiency and repetition of ISDN were combined. NO also controls the activity and expression of AhR by decreasing it (Niedbala et al. 2011). Thus, the final balance between KYN and NO actions on AhR could be a partial reduction of AhR expression after ISDN, leaving enough active AhR to promote the pronociceptive effect observed after its direct activation by ITE. This enhanced pro-inflammatory state could be a potential inducer of pain facilitation and persistence, as observed by the long-lasting CMH in mice after TRP deficiency. It should be noted that TRP deficiency did not affect ISDN-induced extracephalic hypersensitivity since the targeted pathway is of a different nature. This difference could be due to the different mechanisms of both hypersensitivities considering in episodic migraine cephalic hypersensitivity is mostly mechanical, whereas extracephalic is mostly thermal (Guy et al. 2010).

Our hypothesis is that AhR activation promotes the sensitization of the inflammatory signaling pathways, facilitating nociceptive response and central sensitization of the trigeminal system. Understanding the potential pronociceptive nature of AhR was based on the administration of potent agonists and antagonists. The systemic activation of AhR by the non-toxic agonist ITE, prior to ISDN administration, enhanced the intensity and duration of ictal CMH. On the other hand, AhR inhibition by non-toxic TMF prevents both ictal and interictal ISDN-induced CMH. The blockage of AhR by TMF does not allow KYN to execute its function as a ligand, and hence the IDO/AhR axis is potentially hindered. And when we combined TMF with a TRPdeficient diet, that leads to a lower TMF-induced antalgic effect. Through that, we have been able to show that AhR has a pro-nociceptive action during migraine initiation and progression. For future steps within this part of the project , the AhR knockout model will be used to further understand and identify whether the role of the AhRs is peripheral or central.

Triptan Efficacy

Triptans are highly effective if taken early within the first few minutes of the start of the attack, but when the attack is established, and there is the presence of allodynia (indicative of ongoing central sensitization) patients are no longer responsive (Rami Burstein, Collins, and Jakubowski 2004). Although triptans are recommended as a first-line treatment, approximately 30-40 % of migraineurs suffer from insufficient efficacy or tolerability to triptans [START_REF] Lipton | Impact of NSAID and Triptan Use on Developing Chronic Migraine: Results From the American Migraine Prevalence and Prevention (AMPP) Study[END_REF]Viana et al., 2013). In the case of sumatriptan, for example, non-responders show lower and slower absorption after oral administrations compared to responders (A. [START_REF] Ferrari | Interindividual Variability of Oral Sumatriptan Pharmacokinetics and of Clinical Response in Migraine Patients[END_REF]. Moreover, in women, the efficacy rate of triptans is the same as in men, however, their risk of a headache recurrence is higher [START_REF] Casteren | Sex Differences in Response to Triptans: A Systematic Review and Meta-Analysis[END_REF].

Because sumatriptan works at the level of the serotonergic 5-HTR1B/1D receptors, modulations in serotonergic neurotransmission described in migraineurs may influence the functioning of these receptors and hence be the source of triptan inefficacy. Changes in serotonin levels can be produced by rapid dietary reduction of TRP, its precursor, and a powerful inducer of migraine features such as LH and pain (P. D. Drummond 2005).

We demonstrated a lack of efficacy of sumatriptan when it came to both CMH in both sexes and LH in females induced by an acute ISDN administration. Sumatriptan's lack of activity, which is also present in migraineurs, might be attributed to serotoninergic modulations or receptor functioning. Quantification of 5-HTR1B/1D expression reveals that under TRP-deficient circumstances, the receptors' expressions, notably 5-HTR1D, increased significantly, at least at the Sp5C level. This increase in expression, together with low serotonin levels, may lead to migraine episodes (Panconesi 2008).

Furthermore, the decrease in sumatriptan effectiveness may be connected to receptor functioning, whereby it is sensitized rather than expressed for 5HTR1B, for example. This alteration in expression has not been documented in migraine or other pain models.

For the next step in this part of the project, we will perform a functionality assessment of 5-HTR1B/1D receptors using patch clamp. This will give insight into the electrophysiological status of these receptors under TRP-deficient conditions coupled with migraine. Moreover, we will assess the efficacy of another triptan within the same model which will allow us to understand if this lack of efficacy is limited to sumatriptan or affects other triptans as well. Since we only assessed the efficacy of sumatriptan at the level of CMH and LH, we will also assess it on EMH under a TRP deficient diet, to see if the effect is also limited to CMH or not. As for the TRP-AhR axis and its nociceptive role, we have been able to showcase a novel mechanism contributing to the augmentation of cephalic hypersensitivity in migraine-like pain. This allows us to further target AhR as a therapeutic means in the field of nociception. However, multiple sides should be taken into account including the physiological and pathological side effects potentially induced through the action on these receptors. Nevertheless, further research is in store to understand this area.

CONCLUSION

SECONDARY PROJECT Visualizing Neuronal Dynamics of the Posterior Thalamus in a Mouse Model of Migraine-like Pain

During my thesis, I also implemented a new technique in our lab using a miniscope system for in vivo calcium imaging in freely-moving mice (Figure 1). The aim was to investigate the role of the posterior thalamus in the light hypersensitivity (LH) induced by ISDN.

Photophobia or LH is considered the most bothersome symptom reported in 80 % of migraineurs (Choi et al. 2009), during (Lovati et al. 2013) and between headache attacks (Chong, Starling, and Schwedt 2016;Vanagaite et al. 1997). When understating the mechanism by which photophobia arises, four distinct definitions have been accepted: (1) LH is an abnormal sensitivity to light, associated with a reduced photosensitivity threshold, as shown by migraineurs during the interictal period; (2) light can exacerbate or trigger headache; (3) LH is described as a moderate to severe ocular discomfort; and (4) LH is a general aversion to a non-noxious light intensity (Fine and Digre 1995;Lebensohn 1951;[START_REF] Noseda | A Neural Mechanism for Exacerbation of Headache by Light[END_REF]Noseda, Borsook, and Burstein 2017). Despite the research advances in the last years, the exact mechanisms of different modalities of LH are still unclear.

The thalamus is a central relay for nociceptive information from the lower brain regions to different cortical areas via the trigeminovascular pain pathway (Younis et al. 2019). It is a crucial player in the pathophysiology of migraine. However, it is not only a relay but also a potent integration center of light inputs from the retinal ganglion cells (RGC) and then conveys it to the primary visual cortex [START_REF] Noseda | A Neural Mechanism for Exacerbation of Headache by Light[END_REF].

Hence, the thalamus could be an integration center for both light and nociceptive information (Rami Burstein, Collins, and Jakubowski 2004). Indeed, Noseda and his team were able to demonstrate the existence of different subpopulations of trigeminovascular neurons at the level of the posterior thalamus (Po) that are either dura sensitive or that respond to both light and dural stimulations [START_REF] Noseda | A Neural Mechanism for Exacerbation of Headache by Light[END_REF]). However, in Noseda's work, experiments were performed on anesthetized rodents and not under migraine conditions. Stimulation of the Po nuclei was sufficient to induce light-aversive behavior in a recent mouse model of migraine (Sowers et al. 2020).

Based on their findings, this opened a question of how would Po neurons be affected in terms of functionality during a migraine attack and under light stimulations.

Hence we aimed to assess the neuronal activity of these neurons using in vivo calcium imaging in free-moving male and female C57BL/6 mice under migraine-like conditions using single or repeated (three, twice per day for two days) intraperitoneal injections of ISDN (Risordan®, Sanofi-Aventis, Paris, France; 10 mg/kg in a 10 ml/kg). Light stimulations were established at 50 or 300 lux. As shown in Study 1 from this thesis, 50 lux illuminance did not induce LH in ISDN-injected mice, while both sexes were photophobic at 300 lux. In the case of males, light aversion to 300 lux was innate. In Briefly, mice were anesthetized and fixed on a stereotaxic frame. The scalp was removed. The skull on top of the injection area was carefully drilled and removed using a dental drill. Visualization of the neurons at the level of the Po was done using an injection of a ready-to-image AAV1.Camk2a.GCaMP6f.WPRE.bGHpA virus (Inscopix, CA, USA) which was administered using a glass electrode directly at the level of the Po (AP: -2, MD: 1.2, and DV: -3; Figure 1A) (Paxinos and Franklin 2019).

During the same surgical procedure, an integrated micro endoscopic GRIN (Gradient Index) lens (Inscopix, CA, USA) was lowered towards the injection area of the Po. The lens was then secured to the mouse's skull and a cap was placed for protection. A total of 5 males and 3 females were successfully implanted.

Six-week post-surgery, mice were habituated for one week to the testing room with a dummy miniscope placed on their heads. Then, during the testing session, mice were attached to the miniscope (nVoke 2.0, BB-11170203, Inscopix, CA, USA), placed inside the open field, and allowed to move freely for 30 min (Figure 1B). The calcium images were obtained by the nVoke acquisition software (Inscopix, CA, USA; Figure 1C). Baseline measurements of neuronal activity were recorded under the three following conditions: dark (light-OFF), non-aversive light (50 lux), and aversive light (300 lux) each for 40 seconds. ISDN was then administrated, and the same procedure was followed at three-time points post-injection (30, 60, and 180 min). The raw data was then processed with custom-written scripts in Matlab (The MathWorks Inc., 2015), using the subroutine from InscopiX Data Processing software. DF/F and the number of calcium events were used to estimate the cells' synchronicity and activity clusters.

Three main preliminary results were obtained from these experiments:

-One hour after ISDN injection, a significant variation in the activity of Po neurons was observed, concomitant with the peak of cephalic hypersensitivity. Cells that had physiologically increased activity reduced the calcium event, and vice versa, neurons with basal decreased activity increased their responses. However, the global effect could be translated as a reduction of Po neuronal function.

-Light stimulation at 300 lux showed a variation in the activity of the cells, with an overall increase in the calcium response and hence an augmentation of the activity of certain light-sensitive neurons.

-Light stimulation at 300 lux one-hour post-ISDN administration globally increased the calcium activity of Po neurons. Indicating the existence of a subset responding to both pain and light.

-Four different subpopulations of Po neurons were identified, according to their activity after light and/or ISDN stimuli; (1) neurons that-respond to migraine-like pain induced by ISDN, (2) neurons that respond to light (300 lux but not 50 lux), (3) neurons that respond to both nociception and light and (4) non-responder neurons. The proportions of these neurons were different, being neurons that respond to both light and ISDN as the most important subpopulation, followed by ones responding to ISDN alone.

For the moment, the reduced number of recorded neurons from females did not allow us to compare both sexes. For the future steps in this project, more animals are to be added, in addition to a more in-depth analysis of the prior obtained recordings and the ones to come with an assessment of neuronal synchrony and discharge frequency.

Moreover, the next experiments will include combined in vivo calcium dynamics recording with cephalic and extracephalic von Frey stimulations.

Figure 1 -

 1 Figure 1-Stages of a Migraine Attack. ..........................................................................

Figure 2 -

 2 Figure 2-Features Associated with Episodic and Chronic Migraine.. ...........................

Figure 3 -

 3 Figure 3-Triggers and Factors Affecting a Migraine Attack. ........................................

Figure 4 -

 4 Figure 4-Nociceptive Somatosensory Organization of the Orofacial Area ..................

Figure 5 -

 5 Figure 5-Organization of the Sp5c ................................................................................

Figure 6 -

 6 Figure 6-Anatomy of Neurovascular Pathways Modulating the Trigeminal Neurotransmission ..........................................................................................................

Figure 7 -

 7 Figure 7-Neuronal Sensitization of Trigeminovascular system during migraine. ........

Figure 8 -

 8 Figure 8-Synthesis of Noradrenaline .............................................................................

Figure 9 -

 9 Figure 9-The Localization of the LC .............................................................................

Figure 10 -

 10 Figure 10-The Heterogeneous Cellular and Molecular Composition of the LC ...........

Figure 11 -

 11 Figure 11-The Afferent and Efferent Connections of the LC. ......................................

Figure 12 -

 12 Figure 12-The LC in Migraine. .....................................................................................

Figure 13 -

 13 Figure 13-Morphological and Functional Astrocytic Changes After Noxious Stimulation and Nerve Injury. ........................................................................................

Figure 14 -

 14 Figure 14-Major pro-inflammatory glial-mediated alterations to excitatory synapses that contribute to Central Sensitization. .........................................................................

Figure 15 -

 15 Figure 15-The Structure of the Aryl Hydrocarbon Receptor ........................................

Figure 16 -

 16 Figure 16-Aryl Hydrocarbon Receptor Signaling Pathways. ........................................

Figure 17 -

 17 Figure 17-Roles of Aryl Hydrocarbon Receptors .........................................................

Figure 18 -

 18 Figure 18-The Tryptophan/Kynurenine Pathway ..........................................................

Figure 19 -

 19 Figure 19-The Tryptophan/Serotonin Pathway .............................................................

Figure 20 -

 20 Figure 20-The Tryptophan/Indole Pathway ..................................................................

Figure 21 -

 21 Figure 21-Schematic Representation of the Experimental Hypotheses ........................

  2023-Mrad Y et al. " Visualizing neuronal dynamics of the posterior thalamus in a mouse model of migraine " (Clermont-Ferrand, Doctoral School Day) 2022-Mrad Y et al. " Aryl hydrocarbon receptors are involved in the pathogenesis of migraine " (Vienna, 16th European Headache Congress) 2022-Mrad Y et al. " L'alimentation a-t-elle un rôle dans la chronicisation de la migraine?" (Lille, French Society for the Study and Treatment of Pain 2022) 2022-Mrad Y et al. "Dietary Tryptophan as a Potential Factor of Migraine Chronification" (Clermont-Ferrand, Pain Symposium 2022) CONTRIBUTIONS AND COLLABORATIONS Some of the procedures and techniques performed in this thesis have been carried out with the help and collaboration of other researchers. Their names and respective organizations are listed below. International Collaborations • Pr. Maria Angeles Moro, Dr. María Isabel Cuartero Desviat, and Dr. Alicia García Culebras from the Neurovascular Pathophysiology, Cardiovascular Risk Factor and Brain Function Program, Centro Nacional de Investigaciones Cardiovasculares (CNIC) (Madrid, Spain). • Dr. Igor Horrillo and Mrs. Ines Erkizia Santamaría (Ph.D. student) from the Department of Pharmacology, University of the Basque Country (Biscay, Spain).

Figure 1 -

 1 Figure 1-Stages of a Migraine Attack. Migraine attacks in patients with aura are developed in four distinct phases: prodrome, aura, headache, and postdrome. b, most common symptoms; c, less common symptoms. Adapted from (M. D. Ferrari et al. 2022).

Figure 2 -

 2 Figure 2-Features Associated with Episodic and Chronic Migraine. This representation summarizes the most important triggers and risk factors of migraine. Adapted from (Sheena K. Aurora and Brin 2017).

Figure 3 -

 3 Figure 3-Triggers and Factors Affecting a Migraine Attack. This representation summarizes the most important triggers and risk factors of migraine. Adapted from (Khan et al. 2021).

  . Mechanical, thermal, or chemical stimuli only stimulate when their intensities are in the noxious range. Based on their anatomical and functional characteristics, nociceptive fibers can be classified into three groups: (i) Aα and Aβ myelinated fibers that transmit information at a fast speed (with conduction velocities >30 m/s) to the spinal and medullar dorsal horn laminae III, IV, and V; (ii) Aδ fibers which are lightly myelinated and transmit nociceptive signals slowly (conduction velocities 2.5-3 m/s) to the laminae I and V; and (iii) C unmyelinated fibers have slow transmission (conduction velocities <2.5 m/s) and transmit to laminae I and II (Abraira and Ginty 2013) (Figure

Figure 4 -

 4 Figure 4-Nociceptive Somatosensory Organization of the Orofacial Area. The three divisions of the trigeminal nerve supply a variety of tissues including meninges. The primary afferent neurons mediating nociceptive messages (Aδ and C) project to the spinal tract nucleus. Ascending pathways arise from the spinal tract nucleus to reach higher-order areas such as the thalamus and cortex. Descending pathways can also modulate the activity of the thalamus and spinal nucleus. Oralis subnucleus (Vo), Interpolaris subnucleus (V1), Caudalis subnucleus (Vc), ophthalmic (V1), maxillary (V2), and mandibular (V3).

Figure 5 -

 5 Figure 5-Organization of the Sp5c. The distinct laminae of the Sp5c with the corresponding innervations of the branches of the trigeminal nerve (V1, V2, and V3). Following nociceptive stimuli messages are transmitted through C fibers (Toward laminae I, IIo, and IIi) and Aδ-fibers (Toward laminae I, IIo, and V). Non-nociceptive messages are transmitted by Aβ-fibers (Toward laminae III, IV, and V). Lamina I (I), lamina II outer (IIo), lamina II inner (IIi), lamina III-V (III-V), ophthalmic (V1), maxillary (V2), and mandibular (V3), DRG: Dorsal Root Ganglion. (Created with Biorender.com).

Figure 6 -

 6 Figure 6-Anatomy of Neurovascular Pathways Modulating the Trigeminal Neurotransmission. Schematic representation of ascending and descending neuronal pathways in the medullary and upper cervical dorsal horn, which are involved in aspects of migraine and cluster headache. Au: auditory association cortex; DRG: dorsal root ganglion; Ect: entorhinal cortex; Ins: insula; LC: locus coeruleus;M1/M2: primary and secondary motor cortex; PtA: parietal cortex; PAG: periaqueductal gray; RS: retrosplenial cortex; RVM: rostral ventromedial medulla; S1/S2: primary and secondary somatosensory cortex; SPG: sphenopalatine ganglion; SuS: superior salivatory nucleus; TNC: trigeminal nucleus caudalis; TG: trigeminal ganglion; V1/V2: primary and secondary visual cortex. Adapted from[START_REF] Hoffmann | Neurovascular Mechanisms of Migraine and Cluster Headache[END_REF].

Figure 7 -

 7 Figure 7-Neuronal Sensitization of Trigeminovascular system during migraine. Schematic representation of the peripheral sensitization of the trigeminal ganglion neurons (mediator of periorbital throbbing pain), and the central sensitization of the spinal trigeminal nucleus caudalis (Sp5c) (mediator of cephalic cutaneous hypersensitivity), and thalamic trigeminovascular neurons (responsible of extracephalic cutaneous hypersensitivity) during migraine attack. Adapted from (Noseda and Burstein 2013).

Figure 8 -

 8 Figure 8-Synthesis of Noradrenaline. The rate-limiting enzyme tyrosine hydroxylase converts the amino acid L-tyrosine into L-dihydroxyphenylalanine. L-DOPA is then decarboxylated to become

Figure 9 -

 9 Figure 9-The Localization of the LC. The location and shape of the LC according to its orientation.

Figure 10 -

 10 Figure 10-The Heterogeneous Cellular and Molecular Composition of the LC. (A) Differing locations of the morphologically different LC NE+ cells along the dorsal-ventral axis. (B) Expression of different sets of molecules in NE+ cells, with subsets co-releasing other molecules and expressing different neurotransmitters and receptors. NE: Norepinephrine; ARa1: adrenoceptor a1 subtype; ARa2: adrenoceptor a2 subtype; NPY: Neuropeptide Y; me5: mesencephalic trigeminal nucleus; LC: locus coeruleus. Edited and Adapted from (Schwarz and Luo 2015).

BFigure 11 -

 11 Figure 11-The Afferent and Efferent Connections of the LC. (A) The different projections from brain regions towards the LC, starting with those encoding visceral and sympathetic information from the brainstem and midbrain in black, the emotional, homeostatic, and cognitive information from the forebrain regions in red, and the neuromodulatory inputs in blue. BNST: bed nucleus of the stria terminalis; CeA: central nucleus of the amygdala; DMH/LH: dorsomedial and lateral hypothalamus; DR: dorsal raphe; Gi: nucleus gigantocellularis; IC: insular cortex; LC: Locus coeruleus; mPFC: medial prefrontal cortex; NTS: nucleus tractus solitarius; PAG: periaqueductal gray; PGi: nucleus

Figure 12 -

 12 Figure 12-The LC in Migraine. (A) Nociceptive input from the trigeminal ganglia (TG) is mediated byprimary afferents that synapse centrally on the trigeminocervical complex (TCC). Ascending projections from the TCC mostly target the sensory thalamic nuclei; however, excitatory projections also target the LC, activating it. The LC receives descending excitatory inputs from the hypothalamus during wakefulness, which enhances its activity (green arrow). The LC, in turn, sends noradrenergic projections to the majority of the CNS, including descending projections to the TCC and spinal cord as well as ascending projections to the thalamus and cortex, which play roles in arousal and nociceptive processing. (B) Loss of LC noradrenergic signaling impairs trigeminal nociceptive signaling at the cellular level. Adapted from (Vila-Pueyo et al. 2019).

Figure 13 -

 13 Figure 13-Morphological and Functional Astrocytic Changes After Noxious Stimulation and Nerve Injury. Based on the phenotypic changes of astrocytes, they can be divided into reactive and scarforming astrocytes. Reactive astrocytes can be further classified by A1 and A2 subtypes. GFAP: Glial fibrillary acidic protein; Cdh2: Cadherin-2; Sox9: SRY-Box Transcription Factor 9; CSPG: Chondroitin Sulfate Proteoglycan 4; C3: Complement 3; CFB: Complement factor B; MX1: myxovirus resistance-1.

  Changes in the expression of cell surface receptors, certain polarization reactions, and the release of several inflammatory mediators are all linked to microglial activation and can either play a beneficial neuroprotective role in tissue repair or a harmful neurotoxic response[START_REF] Wendimu | Microglia Phenotypes in Aging and Neurodegenerative Diseases[END_REF]. A traditionally activated M1like phenotype or an alternatively activated M2-like phenotype are two broad polarization states that are typically used to characterize microglial activity. The triggering stimuli, phenotypic marker expression, and mediator secretion of these polarization states vary, which all affect the outcome. Generally speaking, proinflammatory and neurotoxic responses are linked to the classically activated M1 phenotype, whereas anti-inflammatory and neuroprotective actions are mostly mediated by the M2 phenotype[START_REF] Tang | Differential Roles of M1 and M2 Microglia in Neurodegenerative Diseases[END_REF]. Interferon (IFN) and/or the Gramnegative bacterial endotoxin lipopolysaccharide both promote the M1-like microglia phenotype in vitro. M1-like microglia are crucial for inducing the adaptive immune response and innate immunological responses to fight invading invaders(Lehnardt 2010). However, neuroinflammation, oxidative stress, and neurotoxicity are all exacerbated by prolonged activation under pathological situations(Lehnardt 2010; G. J. Song and Suk 2017). Through the release of neurotrophic and anti-inflammatory substances (including IL-4 and IL-10), M2 polarized microglia are frequently linked to tasks like immunological resolution and tissue repair. They may also enter an "alternatively activated" or "acquired deactivation" state. A common link between microglia ad astrocytes exists, whereby by releasing Il-1α, TNF, and C1q cytokines (essential for A1 astrocytes), activated microglia induce the transformation of naïve astrocytes into A1 astrocytes(Liddelow et al. 2017).

Figure 14 -

 14 Figure 14-Major pro-inflammatory glial-mediated alterations to excitatory synapses that contribute to Central Sensitization. Noxious activation of glial cells can lead to the aberrant release of pro-inflammatory mediators, such as TNFα and IL-1β. The overarching effect on excitatory synapses contributes to central sensitization and facilitates the transmission of nociceptive signals to the brain.Some of the major known adaptations include the increased release of glutamate from presynaptic terminals; suppression of astrocytic glutamate reuptake via downregulation of GLT-1 and GLAST activity; release of glutamate from astrocytes, which increases the neuronal excitability, and of D-serine that enhances Ca 2+ influx on postsynaptic neurons. Astrocytic release of ATP also increases postsynaptic excitability via the activation of P2X4R and P2X7R, while IL-1β, TNFα, IFNγ, and CCL2 increase NMDA receptor-mediated excitatory signaling. ERK, extracellular signal-regulated kinase; IFN, interferon; IL, interleukin; TNFα, tumor necrosis factor-α. Adapted from[START_REF] Dodds | Glial Contributions to Visceral Pain: Implications for Disease Etiology and the Female Predominance of Persistent Pain[END_REF]).

Murine

  AhR is encoded by a gene located on chromosome 12, while human AhR is located on chromosome 7. The mouse AhR protein consists of 805 amino acids and its molecular weight is 90 kDa. Its structure consists of an amino (N-) terminal bHLH domain, a PAS domain in the center made up of two degenerate repeats (A and B), and a carboxy (C-) terminal transactivation domain (Figure 15). The N-terminal bHLH domain helps in dimerization and forms the DNA-binding domain. PAS domains control DNA recognition, chaperone interactions, and binding of ligands. In addition, the PAS coupled with the N-terminal bHLH domain, are supporters of the heterodimerization of AhR with ARNT. The transactivation domain can be divided into a first part rich in acid residues, a second part rich in glutamine necessary for the binding of cofactors, and a final part rich in proline/serine/threonine. The sequence of the transactivation domain is very different between mice and humans with only 58% homology. This difference raises various hypotheses such as the divergence in the coactivator recruitment or the different levels of gene induction.

Figure 15 -

 15 Figure 15-The Structure of the Aryl Hydrocarbon Receptor. N: Amino-terminal; bHLH: basic helixloop-helix; PAS A/PAS B: period aryl hydrocarbon receptor nuclear translocator single-minded A/B; Qrich: Glutamine Rich; C: Carboxy-terminal; Hsp90: heat shock protein 90. Adapted from (Lin, Dai, and Xia 2022).

Figure 16 -

 16 Figure 16-Aryl Hydrocarbon Receptor Signaling Pathways. Upon binding of the ligand, the complex dissociates activating multiple pathways (the genomic pathway and pathways regulating the transcription of target genes). Other parts of the complex such as c-Src trigger different pathways, activating FAK or MAPK with modulation of proliferation and adhesion. AIP: AhR-interacting protein; AhR: aryl hydrocarbon receptor; ARNT: aryl hydrocarbon receptor nuclear translocator; c-Src: cellular-sarcoma; FAK: focal adhesion kinase; Hsp90: heat shock protein 90; MAPK: mitogen-activated protein kinase;NF-κB: nuclear factor-kappa B; XAP2: X-associated protein 2; XME: xenobiotic metabolizing enzyme; XRE: xenobiotic response element. Adapted from[START_REF] Larigot | Aryl Hydrocarbon Receptor and Its Diverse Ligands and Functions: An Exposome Receptor[END_REF]).

  environmental pollutants (xenobiotics) from halogenated aromatic hydrocarbons (e.g. TCDD) and polycyclic aromatic hydrocarbon families. Ligands from the halogenated aromatic hydrocarbons family have a stronger affinity for AhR than polycyclic aromatic hydrocarbons. Most of these xenobiotics cause numerous toxic effects. For example, TCDD exposure in rats reduces food intake, influences body composition, and alters the function of the hypothalamus[START_REF] Lindén | Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) on Hormones of Energy Balance in a TCDD-Sensitive and a TCDD-Resistant Rat Strain[END_REF]. In humans, TCDD induces chronic malformations that manifest with time including, hypertension, diabetes, neural damage, and atherosclerosis[START_REF] Pelclová | Adverse Health Effects in Humans Exposed to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD)[END_REF].Non-xenobiotic exogenous AhR agonists, provided by food, have also been identified. Vegetables are a source of indole-3-acetonitrile, indole-3-carbinol, and 3,3.diindolylmethane (Table2). The resveratrol (3,5,4'trihydroxystilbene), which belongs to the polyphenol family, is present in certain fruits and has recognized antioxidant and anti-inflammatory activity. Several phytochemicals, such as flavones, isoflavones, flavonols, and flavanones also act as AhR modulators[START_REF] Goya-Jorge | Plant Occurring Flavonoids as Modulators of the Aryl Hydrocarbon Receptor[END_REF][START_REF] Xue | Mechanisms and Therapeutic Prospects of Polyphenols as Modulators of the Aryl Hydrocarbon Receptor[END_REF]).

  [START_REF] Abbott | RT-PCR Quantification of AHR, ARNT, GR, and CYP1A1 MRNA in Craniofacial Tissues of Embryonic Mice Exposed to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin and Hydrocortisone[END_REF]) and accelerates the aging of mice[START_REF] Bravo-Ferrer | Lack of the Aryl Hydrocarbon Receptor Accelerates Aging in Mice[END_REF].Disruption of any of the homeostatic functions can disrupt other organs and from there the cascade continues potentially leading to a communicational impairment between organs.

Figure 17 -

 17 Figure 17-Roles of Aryl Hydrocarbon Receptors. AhR are regulators of a plethora of specific processes at the level of different physiological barriers, organs, and tissues. Any form of disturbance or deficiency in the function of this could lead to pathophysiological states. Adapted from (Larigot et al. 2022).

Figure 18 -

 18 Figure 18-The Tryptophan/Kynurenine Pathway. Tryptophan is converted to kynurenine by IDO. Kynurenine is then further catabolized to give either the neuroprotective target kynurenic acid (through the action of KAT) or the neurotoxic one, quinolinic acid (through the action of KMP). IDO: indoleamine 2,3-dioxygenase; KMO: kynurenine-3-monooxygenase; KAT: kynurenine aminotransferase. (Created with BioRender.com)

  , or catabolized to 5-hydroxy indole acetaldehyde by MAO, and from there to 5-hydroxyindoleaceticacid (5-HIAA) by aldehyde dehydrogenase[START_REF] O'mahony | Serotonin, Tryptophan Metabolism and the Brain-Gut-Microbiome Axis[END_REF]. The serotonin receptors, spread out in different areas of the CNS, all of which are GTP binding protein-coupled receptors are classified into seven families, further subdivided into 14 subtypes except the 5HT-3 receptor[START_REF] Nichols | Serotonin Receptors[END_REF]. In terms of function, serotonin is important for mood, pain, appetite, and sleep regulation, in addition to its role in cognition (memory and learning), and feelings of happiness and well-being (L. A.[START_REF] Jones | The Ever-Changing Roles of Serotonin[END_REF]).

Figure 19 -

 19 Figure 19-The Tryptophan/Serotonin Pathway. 1-Tryptophan is converted to serotonin by TPH and AADC enzymes. 2-Serotonin is moved into the vesicles located in the axon terminal through the VMAT2. 3-Following an action potential, serotonin is released into the synapse. 4-Activation of autoreceptors and post-synaptic receptors. 5-Free-5HT is taken from the synapse by the 5-HTT and the rest can be metabolized giving rise to 5-HIAA by MAO and aldehyde dehydrogenase. 5-HTP: 5hydroxytryptophan; 5-HT: serotonin; 5-HTT: serotonin transporter; 5-HIAA: 5-hydroxyindoleaceticacid; AADC: L-aromatic amino acid decarboxylase; MAO: monoamine oxidase; Trp: Tryptophan; TPH: tryptophan hydroxylase; VMAT2: vesicular monoamine transporter isoform 2. Adapted from (Körtési, Spekker, and Vécsei 2022).

Figure 20 -

 20 Figure 20-The Tryptophan/Indole Pathway. At the level of gut microbes; tryptophan is metabolized into indole and other indole derivatives. ArAT: aromatic amino acid aminotransferase; IA: anholocyclic acid; IAA: indole-3-acetic acid; IAAld: indole-3-acetaldehyde; IAld: indole-3-aldehyde; IAM: indole-3acetamide; ILA: indole-3-lactic acid; IPA: indole-3-propionic acid; IPYA: indole-3-pyurvic acid. Edited and adapted from (Roth et al. 2021).

Figure 21 -

 21 Figure 21-Schematic Representation of the Experimental Hypotheses. Migraine could be linked to an abnormal systemic and neuronal inflammatory state, which can increase the risk of spontaneous headache triggering, and hence, contribute to progression from an episodic to a chronic state. Inflammation is modulated by tryptophan-derived metabolites, which are implicated in both episodic and chronic migraine, through the aryl hydrocarbon receptor (AhR). Hence, TRP and neuroinflammation could be key players in migraine chronification, and in the therapeutic efficacy of abortive drugs. (Created with Biorender.com).

  hypersensitivity, but new controlled studies have shown conflicting results (RamiBurstein, Collins, and Jakubowski 2004;[START_REF] Cady | Elevated Saliva Calcitonin Gene-Related Peptide Levels during Acute Migraine Predict Therapeutic Response to Rizatriptan[END_REF]. Differences in pharmacokinetic parameters and bioavailability of sumatriptan (Peer Tfelt-Hansen 2007) or genetic diversity in 5-HTR1B have also been suggested(MaassenVanDenBrink et al. 1998).

including 5 -

 5 HT production(Höglund, Øverli, and Winberg 2019). Dietary TRP has been shown to have an inverse relationship with migraine risk (Razeghi[START_REF] Jahromi | Association of Diet and Headache[END_REF], suggesting that low brain concentrations of TRP itself or its metabolites enhance the risk of a migraine attack(Biringer 2023). Short-term reduction of TRP intake induces nausea, dizziness, and motion sickness (P. D. Drummond 2005), boosts the intensity of headaches and LH in migraineurs (P.Drummond 2006), and modulates the brain availability of 5-HTR1B in healthy individuals(Baldassarri et al. 2020). Hence, we sought to study the contribution of TRP on the abortive effect of sumatriptan in a mouse model of migraine-like pain induced by a single systemic administration of isosorbide dinitrate (ISDN)(Dallel, Descheemaeker, and Luccarini 2018) in which CMH and LH were used as surrogates for headache-associated symptoms.

  reporting of in vivo experiments) guidelines. Experiments were following the ethical guidelines of Directive 2010/63/UE of the European Parliament and the Council and French Decree 2013-118 on the protection of animals used for scientific purposes. Protocols were approved by the French Ministry of Higher Education and Research (Ref. 2020012716162262).

  Normality and homogeneity of variance were determined using Shapiro-Wilk test, and F or Levene tests, respectively. Unpaired Student's t-test was used to compare mean values between two groups with equal variances. Comparisons of more than two means were performed using repeated (RM) or non-repeated 2 or 3-way ANOVA including Geisser-Greenhouse correction to account for possible unequal variances between groups. Post hoc comparisons between groups were analyzed with the Tukey HSD posttest. T-values, F-values, their associated degrees of freedom, and p-values were summarized in Table

FIGURESFigure 1 .

 1 FIGURES

Figure 2 .

 2 Figure 2. Effect of TRP Deficiency on Cephalic Mechanical Sensitivity and Sumatriptan Efficacy. A-Mechanical sensitivity threshold of female (A1) and male (A2) mice in response to von Frey stimulation after ISDN or vehicle administration at one week of balanced (BAL) or TRP-deficient (TRP -) groups. Data are presented as mean ± S.E.M of (n) mice per group. ***p < 0.001 vs. baseline, +++ p < 0.001 vs. BAL-ISDN group by Tukey HSD posttest following RM 3-way ANOVA. B-Scatter plot shows the area under the curve (AUC) of the cephalic sensitivity threshold for both females and males. The horizontal line represents the mean ± S.E.M of (n) mice per group. Symbols represent individual values. **p < 0.01 by Tukey HSD following 2-way ANOVA. C-Sumatriptan or its vehicle was injected 5 min after the ISDN injection in female (C1) and male (C2) mice in BAL and TRP -groups. *p < 0.05, ***p < 0.001 vs. baseline by Tukey HSD following RM 3-way ANOVA. D-Scatter plot shows the AUC of the cephalic sensitivity threshold after sumatriptan treatment. **p < 0.01, ***p < 0.001 by Tukey HSD following 2-way ANOVA.

Figure 3 .

 3 Figure 3. Effect of TRP-Deficiency on Light Sensitivity and Sumatriptan Efficacy. Light aversion index (LAI) obtained from female (A) and male (B) mice in the mEPM test after ISDN or vehicle administration at one week of balanced (BAL) or TRPdeficient (TRP -) diet. Two light stimulations were tested: 50 and 300 lux. Sumatriptan or its vehicle was injected 5 min after ISDN. The horizontal line represents the mean ± S.E.M of (n) mice per group. Symbols correspond to individual values. *p < 0.05, **p < 0.01, ***p < 0.001 by Tukey HSD posttest following 2-way ANOVA.

Figure 4 .

 4 Figure 4. Expression of 5HTR1B/1D in the Sp5C. The effect of ISDN or vehicle administration on (A) 5HT1B and (B) 5HT1D receptor expression in the Sp5C was evaluated by western blot in balanced (BAL) or TRP-deficient (TRP -) groups.Representative blot for each receptor (A1 and B1) with the total protein used for normalization. Scatter plots show the relative intensity of 5HTR1B (A2) and 5HTR1D (B2). The horizontal line represents the mean ± SEM of (n) mice per group (males and females were pooled). Symbols correspond to individual values obtained from the average of 3 replicates. * p < 0.05 by Tukey HSD posttest following 2-way ANOVA.

  Figure S1A1 S1A2 S1B1 S1B2 S2 Female s S2 Males Data represent F values for repeated measures(RM) or non-repeated measures 2 or 3-way ANOVA, and t values for Student t-test, with

  were performed following ARRIVE (Animals in Research: reporting of in vivo experiments) guidelines, and the ethical guidelines of the Directive 2010/63/UE of the European Parliament and the Council and French Decree 2013-118 on the protection of animals used for scientific purposes. Protocols were approved by the French Ministry of Higher Education and Research (Ref. 2020012716162262).

7. 3 .

 3 Real-time PCR LC samples were prepared with TRIzol Reagent (15596026, Thermofisher Scientific, Massachusetts, United States) and mRNA was reverse-transcribed with the highcapacity cDNA Reverse Transcription kit (Applied Biosystems; Carlsbad, CA, USA) according to the manufacturer's protocol. Real-time quantitative PCR assays (RT-PCR) were performed with an Applied Biosystems 7900HT Fast RT-PCR System sequencer detector (4329001, Thermofisher Scientific). The mRNA expression was normalized to that of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and expressed as the fold difference relative to the vehicle-injected group. All primers and mouse probes were from Applied Biosystems (Thermofisher scientific): AhR (Mm00478932_m1), Cyp1a1

  hind paws) was evaluated using the von Frey test. The baseline threshold was established at 1.20 ± 0.01 g in mice subjected to the BAL diet (Figure 2A Left Panel). A single ISDN injection reduced the extracephalic threshold from 1 h post-administration, with recovery as a function of time (Figure 2A Left Panel). The baseline threshold before the ISDN-3 injection was still lower (0.92 ± 0.08 g) than the baseline before ISDN-1, indicating persistent interictal hypersensitivity. ISDN-3 also reduced the mechanical threshold at 1 h post-injection (0.29 ±0.09 g; Figure 2A Left Panel) and was similar to the ictal threshold after ISDN-1. Extracephalic mechanical threshold returned to baseline level after 3 days. The implementation of a TRP-deficient diet shows no difference compared to the BAL groups (Figure 2A Right Panel) as shown by the similar AUC values (Figure 2B).

FIGURESFigure 1 .

 1 FIGURES

Figure 2 .

 2 Figure 2. A-Extracephalic Mechanical Sensitivity threshold of mice in response to von Frey stimulation after one (ISDN-1) or repetitive ISDN (ISDN-3) or vehicle (Veh) administration at one week of balanced (BAL) or TRP-deficient (TRP -) diet groups. Data are presented as mean ± S.E.M of (n) mice per group. ***p < 0.001 vs. baseline by Tukey HSD posttest following RM 3-way ANOVA. B-Area under the curve (AUC) of the effect of ISDN on the extracephalic sensitivity. Data represent mean ± S.E.M of (n) mice per group. Symbols represent individual values. p > 0.05 by Tukey HSD following 2-way ANOVA.

Figure 3 .

 3 Figure 3. A1-Noradrenergic Functions at the Level of the LC. Representative images of tyrosine hydroxylase (TH)-immunoreactive (IR) stained neurons with their ramifications in the LC under a balanced (BAL) diet coupled with one (ISDN-1) or three (ISDN-3) ISDN administrations or vehicle (Veh). Quantitative assessment of the A2-TH positive area and A3-TH-IR cell number. Data represent mean ± S.E.M of (n) mice per group. Symbols represent individual values. *p < 0.05 by Tukey HSD posttest following 2-way ANOVA. B-Relative expression of TH protein in the LC. *p < 0.05 by Tukey HSD following 2-way ANOVA. C-Measurements of noradrenaline (NA) levels in the LC. *p < 0.05 by Tukey HSD following 2-way ANOVA.

Figure 4 .

 4 Figure 4. AhR Function in the Noradrenergic LC cells. A-Representative images of tyrosine hydroxylase (TH)-immunoreactive (IR) and aryl hydrocarbon receptor (AhR) positive stained cells with their merged image in the LC. Zoomed images show the colocalization of AhR at the level of the TH-positive neurons. Quantitative assessment of the B-Percentage (%) of TH-IR cells expressing AhR (AhR + ) was measured under a balanced (BAL) or TRP-deficient (TRP -) diet coupled with one (ISDN-1) or three (ISDN-3) ISDN administrations. Symbols represent individual values. *p < 0.05 and **p < 0.01 by Tukey HSD posttest following 2-way ANOVA. Box plots represent the C1-cell body and C2-nuclear area of TH-IR AhR + cells in the total LC. *p < 0.05 and ***p < 0.001 by Tukey HSD post-hoc test following 2-way ANOVA. D-Assessment of the activity through measurements of the mRNA expression of the AhR. p > 0.05 by Tukey HSD following 2-way ANOVA. Further assessment is done through the quantification of the E-Percentage (%) of nuclear AhR intensity. *p < 0.05, by Tukey HSD post-hoc test following 2-way ANOVA. Quantifications of the mRNA expression of further downstream AhR genes including the F-AhR repressor (AhRR) and G-CYP1A1 and CYP1B1. p > 0.05 by Tukey HSD following 2-way ANOVA.

  Figure 5. Microglial Reactivity State. A-Representative images of tyrosine hydroxylase (TH) and IBA1-immunoreactive (IR)

Figure 6 .

 6 Figure 6. Astrocytic Reactivity State. A-Representative images of tyrosine hydroxylase (TH) and GFAP-immunoreactive (IR) stained cells with their merged image in the LC under a balanced (BAL) diet coupled with one (ISDN-1) or three (ISDN-3) ISDN or vehicle (Veh;) administrations. A zoomed section into the astrocytes stained with GFAP is also shown. Quantitative assessment of B-the GFAP Intensity, Cthe number of astrocytes, and D-the astrocyte body size (µm 2 ) under one week of BAL or TRP-deficient (TRP -) diet. Data represent mean ± S.E.M of (n) mice per group. Symbols represent individual values. *p < 0.05, **p < 0.01 , and ***p < 0.01 by Tukey HSD posttest following 2-way ANOVA.

Figure 7 .

 7 Figure 7. Astrocytic Morphological Changes under TRP deficiency and/or ISDN Administration. A-Representative astrocyte 3D reconstructions in the LC under a balanced (BAL) or TRP-deficient (TRP -) diet coupled with one (ISDN-1) or three (ISDN-3) ISDN or vehicle (Veh) administrations. Quantitative assessment of the B-Fractal Dimension C-number of branches D-average branch length and E-astrocyte area under ISDN-1 or ISDN-3 administrations at one week of BAL or TRP -diets. Data represent mean ± S.E.M of (n) mice per group. *p < 0.05, **p < 0.01, and ***p < 0.01 by Tukey HSD posttest following 2-way ANOVA. F1-Sholl analysis of reconstructed astrocytes was performed by counting the number of intersections crossing concentric circles traced around the soma (radii increasing at 2.0 µm steps). *p < 0.05, **p < 0.01, and ***p < 0.01 by Tukey HSD following 2-way ANOVA. F2-Area under the curve (AUC) of the number of intersections. *p < 0.05 and ***p < 0.001 by Tukey HSD following 2-way ANOVA.

  the neuroinflammatory state in the Sp5C under ISDNinduced migraine-like pain and the contribution of TRP deficiency Assessment of the glial activation state and the AhR activity under ISDNinduced migraine and TRP-deficient diet conditions were also examined at the level of the spinal trigeminal nucleus caudalis (Sp5c) in female mice. The experimental protocol used was the same as that for the LC in Study 2. Measures were taken 1 h after one (ISDN-1) or three ISDN (ISDN-3) injections, and one week after balanced (BAL)

  These results have opened up a new door for future experiments in the plan to assess if this fast CMH recovery after TRP diet supplementation is coupled with a reduction of neuroinflammation at the level of different migraine-related regions including the LC and Sp5c, or normalization of inflammatory responses.

Figure 1 .

 1 Figure 1. Effect of the Supplementation of TRP on Cephalic Sensitivity. A-After one week of a balanced or TRP-deficient diet, three repetitive injections of ISDN (ISDN-3) were administered. Cephalic mechanical sensitivity was measured before (baseline, B), and 2 to 30 days after ISDN. Change of dietary TRP intake (TRP Supp ) was performed 72 hours after the third ISDN administration. B-Mechanical sensitivity threshold in response to von Frey stimulation after ISDN. Symbols represent mean ± S.E.M of (n) mice per group. *p <0.05, ** p <0.01, and *** p <0.001 vs. baseline by Tukey HSD posttest following 2-way ANOVA. C-Area Under the Curve (AUC) of the cephalic sensitivity threshold from 2 to 30 days. * p <0.05 and ** p <0.01 by Tukey HSD following 1-way ANOVA. C-Levels of TRP in the serum. Symbols correspond to individual values. ** p <0.01 by Tukey HSD following 1-way ANOVA.

( 3 )

 3 Chronic dietary TRP deficiency: a. Reduces systemic TRP and 5-HT levels, but not KYN production. b. Blocks the efficacy of sumatriptan to prevent acute ISDN-induced CMH. c. Promotes pro-inflammatory cytokine release in the plasma and increases TH expression in the LC. d. Worsens acute and persistent ISDN-induced CMH.

  Through our results, we can demonstrate for the first time the pivotal contribution of AhRs as pain mediators through their TRP-derived ligands during migraine progression. Moreover, we have shown the importance of serotoninergic modulation and the contribution of TRP and its metabolites on the effectiveness of sumatriptan. The importance of what we have obtained so far sheds light on the potential clinical importance of this work. To start, what we have obtained is that TRPdeficient diets contribute to the inefficacy of sumatriptan opens a door to further understanding sumatriptan resistance in migraineurs. Potentially altering one's diet and augmenting their daily intake of TRP could ameliorate the efficacy of sumatriptan and hence reduce the number of triptan non-responders. Hence, potentially verifying whether triptan non-responders do have dysregulations in the levels of TRP could be of interest. Further steps could be also to combine a supplementation of TRP coupled with triptan administration among patients resistant to them.

  females, LH was induced by a single ISDN injection. Testing was done in an open field (50x50x30 cm high). Experiments were done following the ethical guidelines of Directive 2010/63/UE of the European Parliament and the Council and French Decree 2013-118 on the protection of animals used for scientific purposes. Protocols were approved by the French Ministry of Higher Education and Research (Ref. 2018051617409279).

Figure 1

 1 Figure 1. In vivo Calcium Imaging in Freely-moving Mice. A-Area of virus injection and placement of the lens

•

  Mr. Vidosav Rajcic and Mrs. Anaïs Raoelina (Master 2 Students); Mr. Robin Lioutaud, Mrs. Manon Meunier (Master 1 Students); Mrs. Tamara Lucien, Mrs. Marion Ledevin, Mrs. Stéphanie Meritet, Mr. Guillaume Marsal, and Mr. Rayan Domet (Undergraduate Students).

Table 1 -Diagnostic Criteria for Migraine as Formulated by the HIS

 1

	Without aura	With aura
	≥5 attacks fulfilling the following	≥5 attacks, with at least 2 fulfilling the following
	criteria:	criteria:
	 Headache lasting 4-72 h	 ≥1 of fully reversible aura symptoms:
	 ≥2 of the following characteristics: Unilateral location Pulsating quality Moderate or severe pain Exacerbation with routine activities	Visual Sensory Speech and/or language Motor Brainstem Retinal
	 ≥1 of the following symptoms: Nausea and/or vomiting Photophobia and phonophobia	 ≥3 of the following characteristics: At least one aura spreads gradually ≥5 min ≥2 aura symptoms occur in succession
		Aura symptoms last 5-60 min
		≥1 aura symptom is unilateral
		≥1 aura symptom is positive
		Aura is accompanied, or followed by a headache

Adapted from (IHS 2018).

Table 2 -Selected AhR Agonists and Antagonists

 2

	Agonists	Biochemical pathway
	L-Kynurenine	Tryptophan Metabolism
	Indole-3-aldehyde/IAld	Microbial metabolism
	Indole-3-acetic acid	Microbial metabolism
	Indole-3-acetaldehyde/IAAld	Microbial metabolism
	Tryptamine	Microbial metabolism
	Indoxyl-3-sulfate	Microbial and host metabolism
	2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester/ITE	Generated from tryptophan and cysteine
	Indole-3-acetonitrile	Derived from cruciferous vegetables
	Indole-3-carbinole	Derived from cruciferous vegetables
	3,3'-diindolylmethane	Derived from cruciferous vegetables
	Bilirubin	Heme metabolism
	Antagonists
	6,2',4'-trimethoxyflavone/TMF	Flavonoid
	Quercitrin	Flavonoid
	Galangin	Flavonoid
	BAY-2416964	Polygonum capitatum

Edited and Adapted from

(Rothhammer and Quintana 2019)

.

Table 1

 1 TABLES

	. Diet Composition
		BAL diet	TRP-deficient diet
	Component	Content	Content
	Cereals	60.4%	60.6%
	Vegetal Proteins	14.0%	14.0%
	Vitamins and Minerals	4.8%	4.8%
	Carbohydrates	10.0%	10.0%
	Oils and Fats	10.0%	10.0%
	Amino Acids	0.9%	0.7%
	Arginine	13.0 g/kg	13.0 g/kg
	Cystine	3.2 g/kg	3.2 g/kg
	Glycine	34.1 g/kg	34.1 g/kg
	Lysine	7.6 g/kg	7.6 g/kg
	Methionine	4.1 g/kg	4.1 g/kg
	Tryptophan	2.3 g/kg	0.3 g/kg
	Both balanced (BAL) and TRP-deficient diets were formulated with
	the following basic ingredients: maize, gelatin, corn oil, sucrose, pre-
	mixture of minerals (calcium, phosphorus, sodium, potassium,
	magnesium, manganese, iron, copper, zinc, and chlorine), pre-mixture
	of vitamins (vitamin A, D3, E, K3, B1, B2, B3, B5, B6, B9, B12,
	biotin, and choline), and an amino acid mixture.

Table 2 .

 2

	Summary of Statistical Analyses Corresponding to Figures 2 to 4, and Table 3	p-value DFd] Finteraction [DFn, p-value F[DFn, DFd] p-value F[DFn, DFd] p-value F[DFn, DFd] ANOVA Figure	0.12 F[3,60]=2.6 0.15 FDiet[1,20]=2.2 <0.0001 FISDN[1,20]= 39.7 <0.0001 FTime[3,60]=46.6 RM 3-way 2A1	0.01 F[3,57]=4.4 0.03 FDiet[1,19]=5.2 <0.0001 FISDN[1,19]= 230.2 <0.0001 FTime[3,57]=106.9 RM 3-way 2A2	0.83 F[1,20]<0.1 0.88 FSex[1,20]<0.1 <0.0001 FDiet[1,20]=29.5 2-way 2B	0.09 F[3,48]=2.2 0.006 FDiet[1,16]=9.8 0.40 0.7 FSumatriptan[1,16]= <0.0001 FTime[3,48]=50.7 RM 3-way 2C1	0.0006 F[3,48]=6.8 0.0007 FDiet[1,16]=17.4 0.19 1.9 FSumatriptan[1,16]= <0.0001 FTime[3,48]=65.1 RM 3-way 2C2	0.26 F[1,16]=1.4 0.93 FSex[1,16]<0.1 <0.0001 FDiet[1,16]=45.5 2-way 2D	0.54 F[2,30]=0.6 0.96 FDiet[1,30]<0.1 0.36 FISDN[2,30]=1.1 2-way 3A-50 lux	0.07 F[2,30]=3.0 0.002 FDiet[1,30]=11.5 <0.0001 FISDN[2,30]=27.6 2-way 3A-300 lux	0.83 F[2,30]=0.2 0.80 FDiet[1,30]<0.1 0.54 FISDN[2,30]=0.6 2-way 3B-50 lux	0.99 F[2,30]<0.1 0.89 FDiet[1,30]<0.1 0.83 FISDN[2,30]=0.2 2-way 3B-300 lux	0.74 F[1,20]=0.1 0.52 FDiet[1,20]=0.4 0.69 FISDN[1,20]=0.2 2-way 4A	0.02 F[1,21]=6.0 0.24 FDiet[1,21]=1.5 0.04 FISDN[1,21]=4.5 2-way 4B	Table 3	0.15 F[1,16]=2.3 <0.0001 FDiet[1,16]=40.7 0.045 FISDN[1,16]=4.7 2-way [5-HT]serum	Females	0.08 F[1,16]=3.6 <0.0001 FDiet[1,16]=27.1 0.01 FISDN[1,16]=7.8 2-way [5-HT]serum	Males	0.004 F[1,13]=12.4 <0.0001 FDiet[1,13]=50.5 <0.0001 FISDN[1,13]=38.2 2-way [TRP]serum	Females	0.05 F[1,13]=4.6 <0.0001 FDiet[1,13]=28.1 0.01 FISDN[1,13]=7.9 2-way [TRP]serum	Males	0.63 F[1,19]=0.2 0.25 FDiet[1,19]=1.4 0.29 FISDN[1,19]=1.2 2-way [TRP]Sp5c	Data represent F values for repeated measures (RM) or non-repeated measures 2 or 3-way ANOVA with corresponding degrees of freedom	(DFn, DFd), and p-values. ANOVA factors were designed as followed: Time, for comparisons between different time points; Diet, for	comparisons between balanced (BAL) and TRP-deficient diet (TRP-); ISDN, for comparisons between and ISDN groups; Sumatriptan vehicle	for the effect mice; and Interaction, for comparisons between vehicle and sumatriptan groups; Sex, for comparisons between female male and	on the considered significant. statistically was variable. p <0.05 dependent between factors

Table 3 . Levels of Tryptophan and Serotonin

 3

		Female	Male
	Serum TRP concentration (µM)
	BAL-Vehicle	176.0 ± 16.0 (5) 90.2 ± 10.9 $$ (5)
	BAL-ISDN	66.0 ± 7.5 (4)	50.7 ± 6.8 (4)
	TRP --Vehicle	55.5 ± 5.6 *** (5) 30.1 ± 2.3 *** (4)
	TRP --ISDN	25.3 ± 7.8 (3)	25.6 ± 6.3 (4)
	Serum 5-HT concentration (µM)
	BAL-Vehicle	1.63 ± 0.05 (5)	1.68 ± 0.08 (5)
	BAL-ISDN	1.79 ± 0.05 (5)	1.73 ± 0.05 (5)
	TRP --Vehicle	1.42 ± 0.04 * (5) 1.31 ± 0.03 *** (5)
	TRP --ISDN	1.45 ± 0.03 ### (5) 1.56 ± 0.04 & (5)
	TRP concentration in the Sp5C (nM)
	BAL-Vehicle	3.0 ± 0.3 (5)
	BAL-ISDN	3.6 ± 0.3 (6)
	TRP --Vehicle	2.7 ± 0.5 (6)
	TRP --ISDN	2.9 ± 0.4 (6)

Data represent mean ± SEM of (n) mice per group. Serum levels of tryptophan (TRP) and serotonin (5-HT) in both sexes were measured after one week of a balanced (BAL) or Trp-deficient (TRP -) diet by ELISA. Trp levels in the Sp5C were measured one week after diets by HPLC (males and females were pooled). Samples were collected 1 h after ISDN or vehicle i.p. administration. *p <0.05 and *** p <0.001 vs. BAL-Vehicle group; ### p <0.05 vs. BAL-ISDN group; & p <0.05 vs. TRP --Vehicle group by Tukey's HSD post-hoc test following 2-way ANOVA. $$ p <0.01 female vs. male by Student-t test.

Table 1 . Summary of Statistical Analyses Corresponding to Figures 1 to 7 and Tables 2 and 3 Fig Test F[DFn, DFd] or tDF p-value F[DFn, DFd] p-value F[DFn, DFd] p-value Finteraction [DFn, DFd] p-value

 1 TABLES Data represent t and F values for Student t-Test, Welch's test, and repeated measure (RM

	1B L RM 3-way	FTime[3,60]=29.90	<0.0001 FISDN[1,20]=28.60	<0.0001 FNB-INJ[1,20]=613.6 <0.0001 F[3,60]=7.90	0.0002
	1B R RM 3-way	FTime[3,60]=50.70	<0.0001 FISDN[1,20]=3.10	0.09	FNB-INJ[1,20]=146 <0.0001 F[3,60]=20.00	<0.0001
	1C	RM 3-way	FTime[10,170]=14.40	<0.0001 FISDN[1,17]=20.40	0.0003 FDiet[1,17]=3.80	0.07	F[10,170]=2.20	0.020
	1D1 2-way	FDiet[1,20]=56.30	<0.0001 FNB-INJ[1,20]=4.50	0.05			F[1,20]=3.20	0.09
	1D2 Student t-Test t[5,5]=5.50	0.08
	1E	Welch's Test t[5,5]=12.3	0.02
	1F1 RM 3-way	FTime[1.9,30.4]=30.60	<0.0001 FTreatment[1,16]=15.72 0.001 FNB-INJ[1,16]=10.30 0.006	F[3,48]=2.24	0.10
	1F2 2-way	FTreatment[1,16]=0.15	0.70	FNB-INJ[1,16]=8.65	0.001			F[1,16]=5.54	0.03
	1G1 2-way	FTime[3,24]=2.50	0.09	FTreatment[3,24]=48.2 <0.0001			F[8,24]=1.10	0.40
	1G2 Student t-Test t[4,4]=1.60	0.006
	2A L RM 3-way	FTime[2.40,38.46]=147.6	<0.0001 FISDN[1,16]=33.32	<0.0001 FNB-INJ[1,20]=3.84 0.06	F[3,48]=0.81	0.49
	2A R RM 3-way	FTime[2.05,32.82]=219.9	<0.0001 FISDN[1,16]=61.89	<0.0001 FNB-INJ[1,16]=0.08 0.79	F[3,48]=4.40	0.008
	2B	2-way	FDiet[1,16]=0.09	0.77	FNB-INJ[1,16]=7.97	0.002			F[1,16]=0.94	0.41
	3A2 2-way	FDiet[1,31]=0.09	0.77	FISDN[2,31]=7.97	0.002			F[2,31]=0.94	0.41
	3A3 2-way	FDiet[1,47]= 0.14	0.71	FISDN[2,47]=1.21	0.31			F[2,47]=0.42	0.66
	3B	2-way	FDiet[1,30]= 13.81	0.0008	FISDN[2,30]=0.14	0.87			F[2,30]=0.17	0.85
	3C	2-way	FDiet[1,28]=11.37	0.002	FISDN[2,28]=2.42	0.11			F[2,28]=3.72	0.04
	4B	2-way	FDiet[1,48]=2.91	0.09	FISDN[2,48]=1.33	0.27			F[2,48]=6.96	0.002
	4C1 2-way	FDiet[1,964]=0.15	0.69	FISDN[2,964]=12.45 <0.0001			F[2,48]=9.15	0.0001
	4C2 2-way	FDiet[1,964]=13.72	0.0002	FISDN[2,964]=9.50	<0.0001			F[2,48]=10.65	0.0001
	4D	2-way	FDiet[1,30]=0.75	0.39	FISDN[2,30]=5.47	0.009			F[2,30]=0.21	0.81
	4E	2-way	FDiet[1,964]=3.30	0.07	FISDN[2,964]=3.56	0.03			F[2,964]=1.31	0.27
	4F	2-way	FDiet[1,30]=1.92	0.18	FISDN[2,30]=3.52	0.04			F[2,30]=0.56	0.57
	4G L 2-way	FDiet[1,29]=0.003	0.96	FISDN[2,29]=1.83	0.18			F[2,29]=0.18	0.83
	4G R 2-way	FDiet[1,31]=0.24	0.63	FISDN[2,31]=3.92	0.03			F[2,31]=2.03	0.15
	5B	2-way	FDiet[1,28]=0.09	0.76	FISDN[2,28]=2.76	0.08			F[2,28]=0.08	0.93
	5C	2-way	FDiet[1,28]=3.29	0.08	FISDN[2,28]=4.06	0.03			F[2,28]=11.43	0.0002
	6B	2-way	FDiet[1,31]=1.25	0.27	FISDN[2,31]=23.30	<0.0001			F[2,31]=0.13	0.88
	6C	2-way	FDiet[1,31]=0.42	0.52	FISDN[2,31]=0.12	0.89			F[2,31]=0.22	0.80
	6D	2-way	FDiet[1,31]=0.65	0.42	FISDN[2,31]=10.41	0.0003			F[2,31]=2.09	0.14
	7B	2-way	FDiet[1,160]=29.05	<0.0001 FISDN[2,160]=16.83 <0.0001			F[2,160]=5.56	0.005
	7C	2-way	FDiet[1,160]=9.31	0.003	FISDN[2,160]=10.40 <0.0001			F[2,160]=2.81	0.06
	7D	2-way	FDiet[1,160]=3.03	0.08	FISDN[2,160]=21.83 <0.0001			F[2,160]=0.88	0.42
	7E	2-way	FDiet[1,160]=0.73	0.39	FISDN[2,160]=47.60 <0.0001			F[2,160]=0.62	0.54
	7G1L 2-way	FDistance[3.5,354.9]=178.00 <0.0001 FISDN[2,101]=8.91	0.0003			F[24,1212]=13.2	<0.0001
	7G1R 2-way	FDistance[3.18,260.5]=145.40 <0.0001 FISDN[2,82]=5.95	0.004			F[24,984]=3.03	<0.0001
	7G2 2-way	FDiet[1,177]=0.90	0.34	FISDN[2,177]=12.68 <0.0001			F[2,177]=2.68	0.07
	Table 2-Plasma
	TRP 2-way	FDiet[1,26]=41.04	<0.0001 FISDN[2,26]=6.15	0.007			F[2,26]=1.75	0.19
	KYN 2-way	FDiet[1,25]=11.25	0.003	FISDN[2,25]=3.70	0.04			F[2,25]=0.43	0.66
	Ratio 2-way	FDiet[1,30]=20.94	<0.0001 FISDN[2,30]=0.64	0.53			F[2,30]=1.25	0.30
	Table 2-LC
	TRP 2-way	FDiet[1,30]=59.5	<0.0001 FISDN[2,30]=0.64	0.53			F[2,30]=3.37	0.05
	KYN 2-way	FDiet[1,28]=0.28	0.60	FISDN[2,28]=0.04	0.96			F[2,28]=5.06	0.01
	Ratio 2-way	FDiet[1,25]=1.92	0.18	FISDN[2,25]=0.14	0.87			F[2,25]=0.11	0.90
	Table 3
	IL-6 2-way	FDiet[1,38]=16.30	0.0003	FISDN[2,38]=11.22	0.0001			F[2,38]=17.79	<0.0001
	IFNg 2-way	FDiet[1,38]=3.48	0.07	FISDN[2,38]=2.51	0.09			F[2,38]=0.54	0.59
	TNFα 2-way	FDiet[1,38]=8.45	0.006	FISDN[2,38]=17.35	<0.0001			F[2,38]=5.63	0.007
	IL1β 2-way	FDiet[1,38]=25.69	<0.0001 FISDN[2,38]=28.99	<0.0001			F[2,38]=29.48	<0.0001

Table 2 . Levels of Tryptophan (TRP) and Kynurenine (KYN)

 2

		Vehicle	ISDN-1	ISDN-3
	[TRP]plasma (nM)
	BAL diet	27.72 ± 1.41 20.52 ± 3.06 17.62 ± 2.5 #
	TRP -diet	5.81 ± 0.73 *** 7.84 ± 2.22 *** 11.01 ± 2.61 ***
	[KYN]plasma (µM)
	BAL diet	1.63 ± 0.20	0.84 ± 0.14	1.39 ± 0.24
	TRP -diet	1.01 ± 0.11	1.80 ± 0.53	1.37 ± 0.13
	[KYN]/ [TRP] Ratioplasma
	BAL diet	85.56 ± 26.09 67.33 ± 15.56 134.85 ± 28.65
	TRP -diet	209.78 ± 29.72 * 193.01 ± 19.56 + 186.27 ± 36.42
	[TRP]LC (µM)
	BAL diet	2.55 ± 0.17	3.08 ± 0.07	3.12 ± 0.25
	TRP -diet	1.69 ± 0.17 *	2.42 ± 0.12	1.81 ± 0.22 +++
	[KYN]LC (nM)
	BAL diet	119.72 ± 19.47 174.91 ± 8.28 128.82 ± 13.87
	TRP -diet	78.60 ± 16.37 112.44 ± 17.53 96.03 ± 21.44
	[KYN]/ [TRP] RatioLC
	BAL diet	48.45 ± 9.69	56.21 ± 2.91	50.79 ± 9.51
	TRP -diet	42.65 ± 8.32	43.24 ± 7.00	42.84 ± 6.50
	Data represent mean ± SEM of (5-6) mice per group. Levels of TRP
	and KYN were measured after one week of a balanced (BAL) or
	Trp-deficient (TRP -) diet by HPLC. Samples were collected 1 h
	after ISDN/vehicle administration. *p <0.05 and ***p <0.001 vs.
	corresponding BAL group; # p <0.05 vs. BAL-vehicle group; + p
	<0.05, +++ p <0.001 vs. BAL-ISDN group by Tukey's HSD posttest
	following 2-way ANOVA.

Table 3 . Levels of Pro-inflammatory Cytokines In the Plasma

 3 Data represent F values for repeated measure (RM) or not 2 or 3-way ANOVA with corresponding degrees of freedom (DFn, DFd), and p-values. ANOVA factors were designed as followed: Time, for comparisons between different time points; Treatment, for comparisons between TMF or ITE; Diet, for comparisons between BAL and TRP -; ISDN, for comparisons between ISDN and Vehicle; Interaction for effect between two or three factors on the dependent variable. p <0.05 was considered statistically significant. R: Rostral; M: Middle; C: Caudal; D2: Day 2; D30: Day 30.

		Vehicle	ISDN-1	ISDN-3
	[IL-6] (pg/mL)
	BAL diet	46.76 ± 3.91	95.40 ± 3.54 ***	89.98 ± 3.08 ***
	TRP -diet	96.77 ± 11.17 *** 89.98 ± 90.34 *** 90.34 ± 0.99 ***
	[TNFα] (pg/mL)
	BAL diet	257.82 ± 15.21	447.50 ± 11.70 *** 439.45 ± 11.24 ***
	TRP -diet	400.33 ± 52.71 *** 456.25 ± 21.16 *** 445.83 ± 8.57 ***
	[IL-1β] (pg/mL)
	BAL diet	26.75 ± 2.09	67.28 ± 2.84 ***	76.55 ± 6.24 ***
	TRP -diet	70.72 ± 1.75 ***	70.43 ± 1.18 ***	70.56 ± 1.14 ***
	[IFNγ] (pg/mL)
	BAL diet	208.27 ± 3.39	201.46 ± 1.84	201.45 ± 2.19
	TRP -diet	201.91 ± 3.75	199.41 ± 1.34	199.41 ± 1.38
	Data represent mean ± SEM of (6-8) mice per group. Levels of cytokines
	were measured after one week of a balanced (BAL) or Trp-deficient
	(TRP -) diet by ELISA. Samples were collected 1 h after ISDN/vehicle
	administration. ***p <0.001 vs. corresponding BAL-Vehicle group; by
	Tukey's HSD posttest following 2-way ANOVA.

Table S2 . Effect of ISDN and a TRP-deficient Diet on Noradrenergic LC Cells

 S2

		Group	Rostral	Middle	Caudal
	TH positive area BAL Vehicle 20.05 ± 1.76 17.96 ± 1.84 9.26 ± 1.04
	(10 4 µm 2 )	BAL ISDN-1 28.21 ± 1.33 23.97 ± 1.57 11.12 ± 1.87
		BAL ISDN-3 28.98 ± 3.15 27.67 ± 3.45 14.67 ± 3.16
		TRP -Vehicle 24.33 ± 3.99 19.82 ± 1.85 12.40 ± 1.39
		TRP -ISDN-1 29.11 ± 1.72 19.76 ± 3.06 11.77 ± 1.41
		TRP -ISDN-3 33.44 ± 1.10 21.16 ± 1.56 13.05 ± 1.41
	TH-IR cell	BAL Vehicle 8.33 ± 0.92	13.15 ± 1.04 10.67 ± 1.02
	number/10 4 µm 2 BAL ISDN-1 11.35 ± 1.31 10.73 ± 1.69 8.29 ± 0.94
		BAL ISDN-3 7.38 ± 0.93	12.81 ± 1.04 12.50 ± 1.33
		TRP -Vehicle 8.08 ± 1.18	12.17 ± 1.19 10.07 ± 0.70
		TRP -ISDN-1 7.86 ± 0.77	12.04 ± 0.74 9.89 ± 1.11
		TRP -ISDN-3 7.01 ± 1.22	11.23 ± 1.29 10.60 ± 1.20
	Data represent mean ± SEM of (4-10) animals of the TH positive area and the
	number of TH-IR (immunoreactive) cells under balanced (BAL) or TRP-deficient
	(TRP -) diets coupled with a vehicle, one (ISDN-1), or three ISDN (ISDN-3)
	injections. p >0.05 by Tukey's HSD posttest following 2-way ANOVA.

Table S3 . Morphological Characteristics of AhR + TH-IR Cells

 S3

		Group	Rostral	Middle	Caudal
	% of TH-IR cell	BAL Vehicle	45.52 ± 3.50	51.69 ± 2.81	51.34 ± 2.51
		BAL ISDN-1	46.73 ± 4.05	51.27 ± 3.04	48.11 ± 2.73
	expressing AhR	BAL ISDN-3	59.15 ± 2.85	57.84 ± 2.81	59.97 ± 2.87
		TRP -Vehicle	52.42 ± 2.39	53.86 ± 2.98	51.11 ± 3.94
		TRP -ISDN-1	51.77 ± 1.51	52.50 ± 1.65	54.55 ± 1.19
		TRP -ISDN-3	46.00 ± 4.2	46.08 ± 5.56	50.05 ± 4.38
	Nuclear area (µm 2 ) BAL Vehicle	46.13 ± 1.73	47.08 ± 2.05	50.40 ± 2.00
		BAL ISDN-1	37.42 ± 2.12	46.97 ± 2.15	42.69 ± 2.16
		BAL ISDN-3	48.65 ± 3.52	64.18 ± 3.11 ***	61.22 ± 4.03 *
		TRP -Vehicle	40.90 ± 2.83	46.64 ± 2.15	43.12 ± 1.73
		TRP -ISDN-1	45.93 ± 2.68	47.55 ± 2.01	43.52 ± 2.36
		TRP -ISDN-3	46.39 ± 3.62	42.92 ± 3.80 +++ 47.49 ± 2.26 +
	Cell Body	BAL Vehicle	178.53 ± 5.99	147.23 ± 5.74	156.13 ± 5.62
	(µm 2 )	BAL ISDN-1 BAL ISDN-3	172.00 ± 6.11 188.72 ± 13.00	150.79 ± 5.60 187.58 ± 9.73 ** 192.10 ± 9.35 ** 142.64 ± 6.44
		TRP -Vehicle	155.84 ± 6.44	166.17 ± 5.63	143.16 ± 4.76
		TRP -ISDN-1	182.94 ± 9.38	170.20 ± 6.76	165.32 ± 9.67
		TRP -ISDN-3	197.41 ± 16.06	156.34 ± 6.51	158.34 ± 6.75
	% of AhRNuc	BAL Vehicle	27.58 ± 1.11	31.35 ± 1.15	37.80 ± 1.53
	intensity	BAL ISDN-1	32.92 ± 1.31	32.24 ± 0.86	29.59 ± 1.10 ***
		BAL ISDN-3	22.91 ± 1.50	29.68 ± 1.47	30.80 ± 1.88 *
		TRP -Vehicle	29.59 ± 1.64	27.65 ± 1.52	31.82 ± 1.00 **
		TRP -ISDN-1	28.61 ± 1.03	29.80 ± 0.74	29.86 ± 1.43
		TRP -ISDN-3	24.57 ± 2.00	30.82 ± 2.60	31.21 ± 2.13
	Data represent mean ± SEM of 30-40 cells per animal for 5 animals from the rostrocaudal
	regions of the LC, under balanced (BAL) or TRP-deficient (TRP -) diets coupled with vehicle,
	one (ISDN-1), or three ISDN (ISDN-3) injections. *p <0.05, **p <0.01, ***p <0.001 vs.
	vehicle BAL; + p <0.05, +++ p <0.001 vs. opposite group by Tukey's HSD post-hoc test
	following 2-way ANOVA.

Table S4 . Effect of ISDN and TRP-deficient Diet on the Microglia in the LC

 S4 Data represent mean ± SEM of 3-10 slides per animal from (5-8) mice per group along the rostrocaudal axis of the LC and under balanced (BAL) or TRP-deficient (TRP -) diets coupled with vehicle, one (ISDN-1), or three ISDN (ISDN-3) injections. # p <0.05; ## p <0.01 vs. TRP - vehicle group; + p <0.05; ++ p <0.01; +++ p <0.001 vs. opposite group by Tukey's HSD post-hoc test following 2-way ANOVA.

		Group	Rostral	Middle	Caudal
	Density	BAL Vehicle	2.04 ± 0.31	2.30 ± 0.12	1.88 ± 0.22
	(cell number/10 4 µm 2 )	BAL ISDN-1	1.68 ± 0.04	1.95 ± 0.04	1.89 ± 0.10
		BAL ISDN-3	1.86 ± 0.29	1.98 ± 0.06	2.33 ± 0.12
		TRP -Vehicle	1.94 ± 0.31	2.00 ± 0.12	2.42 ± 0.08
		TRP -ISDN-1	1.62 ± 0.23	1.64 ± 0.02	1.95 ± 0.17
		TRP -ISDN-3	2.00 ± 0.09	2.12 ± 0.05	2.17 ± 0.11
	Average microglia	BAL Vehicle	97.29 ± 3.56 90.45 ± 1.57	95.58 ± 4.12
	size (µm 2 )	BAL ISDN-1	87.73 ± 2.5	91.24 ± 4.31	96.18 ± 3.17
		BAL ISDN-3	96.19 ± 7.24 93.37 ± 5.14	87.64 ± 2.94
		TRP -Vehicle	91.90 ± 6.53 88.00 ± 1.87	91.46 ± 2.80
		TRP -ISDN-1	90.45 ± 4.94 87.68 ± 4.83	90.45 ± 3.35
		TRP -ISDN-3	114.28 ± 6.16 110.27 ± 3.00 +### 106.87 ± 3.21 ++##

Table S5 . Effect of ISDN and TRP-deficient diet on Astrocytes in the LC

 S5

		Group	Rostral	Middle	Caudal
	GFAP Intensity	BAL Vehicle	2.44 ± 0.39 2.07 ± 0.43	1.84 ± 0.30
	(arb unit x10 7 )	BAL ISDN-1	5.76 ± 0.37 ** 4.70 ± 0.81 *	3.01 ± 0.33
		BAL ISDN-3	5.37 ± 0.39 ** 4.38 ± 0.40 *	2.84 ± 0.29
		TRP -Vehicle	3.54 ± 0.65 2.45 ± 0.37	2.69 ± 0.58
		TRP -ISDN-1	5.96 ± 0.51 5.54 ± 0.66 ## 3.4 ± 0.50
		TRP -ISDN-3	5.25 ± 1.00 4.32 ± 0.52	2.83 ± 0.56
	Number of	BAL Vehicle	6.93 ± 0.55 6.85 ± 0.63	7.03 ± 0.41
	astrocytes/100 nm 2	BAL ISDN-1	6.76 ± 0.85 7.28 ± 0.94	6.86 ± 0.60
		BAL ISDN-3	5.80 ± 1.08 6.57 ± 1.17	6.45 ± 0.90
		TRP -Vehicle	8.48 ± 1.01 7.41 ± 1.52	6.64 ± 0.39
		TRP -ISDN-1	6.71 ± 1.03 7.54 ± 0.41	7.04 ± 0.53
		TRP -ISDN-3	6.39 ± 1.28 6.89 ± 1.85	7.61 ± 0.74
	Average	BAL Vehicle	30.51 ± 1.93 29.10 ± 1.80 30.19 ± 1.18
	size (µm 2 )	BAL ISDN-1	38.39 ± 1.66 34.60 ± 0.94 35.00 ± 2.33
		BAL ISDN-3	41.39 ± 0.70 ** 39.75 ± 1.40 * 39.56 ± 2.14 *
		TRP -Vehicle	36.47 ± 3.58 40.56 ± 2.40 + 31.85 ± 0.84
		TRP -ISDN-1	38.37 ± 2.74 32.53 ± 1.51 32.36 ± 1.11
		TRP -ISDN-3	44.96 ± 3.46 41.64 ± 3.22 37.37 ± 3.56
	Data represent mean ± SEM of 3-10 slides per animal from (5-8) mice per group along
	the rostrocaudal axis of the LC and under balanced (BAL) or TRP-deficient (TRP -) diets
	coupled with vehicle, one (ISDN-1), or three ISDN (ISDN-3) injections. *p <0.05 and

**p <0.01 vs. BAL vehicle group; ## p <0.01 vs. TRP -vehicle group; + p <0.05 vs. opposite group by Tukey's HSD post-hoc test following 2-way ANOVA.

Table S6 . Morphometric Analysis of LC Astrocytes

 S6

		Group	Rostral	Middle	Caudal
	Fractal Dimension	BAL Vehicle 1.11 ± 0.01	1.1 ± 0.01	1.13 ± 0.01
		BAL ISDN-1 1.08 ± 0.01	1.11 ± 0.02	1.13 ± 0.03
		BAL ISDN-3 1.05 ± 0.01 *** 1.06 ± 0.01	1.06 ± 0.01 **
		TRP -Vehicle 1.09 ± 0.01	1.06 ± 0.01	1.06 ± 0.01 **
		TRP -ISDN-1 1.04 ± 0.01 #	1.07 ± 0.02	1.06 ± 0.02 ++
		TRP -ISDN-3 1.04 ± 0.01 ##	1.06 ± 0.01	1.06 ± 0.01
	Average Branch length (μm) BAL Vehicle 11.15 ± 0.51	11.61 ± 0.64 10.21 ± 0.73
		BAL ISDN-1 9.04 ± 0.55	10.06 ± 1.16 8.24 ± 0.75
		BAL ISDN-3 13.15 ± 1.04	12.6 ± 0.61	13.66 ± 1.03
		TRP -Vehicle 11.52 ± 0.72	14.1 ± 0.96	12.24 ± 1.31
		TRP -ISDN-1 10.11 ± 0.92	8.75 ± 0.59 ## 10.6 ± 0.97
		TRP -ISDN-3 14.7 ± 0.69 #	12.51 ± 0.91 111.95 ± 1.10
	Number of Branches	BAL Vehicle 16.17 ± 1.12	18.33 ± 2.01 21.83 ± 1.38
		BAL ISDN-1 16.67 ± 1.67	14 ± 1.34	11.17 ± 1.49 ***
		BAL ISDN-3 12.86 ± 1.6	14.3 ± 1.39	15.1 ± 0.97 **
		TRP -Vehicle 19.25 ± 1.13	11.75 ± 0.86 * 12.67 ± 0.76 ***
		TRP -ISDN-1 10.67 ± 0.89 ## 13.83 ± 1.49 9.67 ± 1.31
		TRP -ISDN-3 13.83 ± 1.24 # 17.43 ± 1.27 12.33 ± 1.35
	Astrocyte area (10 3 μm 2 )	BAL Vehicle 0.90 ± 0.10	0.99 ± 0.08	1.06 ± 0.11
		BAL ISDN-1 0.73 ± 0.08	0.62 ± 0.09	0.41 ± 0.12
		BAL ISDN-3 1.42 ± 0.22	1.26 ± 0.14	1.55 ± 0.16
		TRP -Vehicle 1.31 ± 0.10	1.15 ± 0.13	0.94 ± 0.17
		TRP -ISDN-1 0.64 ± 0.13	0.62 ± 0.10 # 0.49 ± 0.04
		TRP -ISDN-3 1.56 ± 0.18	1.46 ± 0.12	1.28 ± 0.16
	Data represent mean ± SEM of 3-10 slides per animal from (3-5) mice per group along the
	rostrocaudal axis of the LC and under balanced (BAL) or TRP-deficient (TRP -) diets coupled
	with vehicle, one (ISDN-1), or three ISDN (ISDN-3) injections. *p <0.05, **p <0.01, ***p
	<0.001 vs. BAL vehicle;

# p <0.05 and ## p <0.01 vs. TRP -vehicle; ++ p <0.01 vs. opposite group by Tukey's HSD post-hoc test following 2-way ANOVA.

Table 1 . GFAP Intensity in the Sp5c

 1

		Vehicle	ISDN-3
	Total
	BAL diet	2.24 ± 0.22	2.57 ± 0.33
	TRP -diet	1.59 ± 0.16	1.88 ± 0.06
	V1
	BAL diet	1.92 ± 0.24	1.91 ± 0.30
	TRP -diet	1.53 ± 0.13	1.38 ± 0.12
	V2
	BAL diet	2.15 ± 0.24	2.44 ± 0.32
	TRP -diet	1.37 ± 0.16	1.67 ± 0.12
	V3
	BAL diet	2.64 ± 0.28	3.36 ± 0.51
	TRP -diet	1.86 ± 0.33	2.60 ± 0.22
	Data represent mean ± SEM of (3-5) Sp5C sections from
	(5-6) mice per group. p >0.05 by Tukey's HSD post-hoc
	test following 2-way ANOVA.

Table 2 . Microglial Density (cell number/10 4 µm2) in the

 2

	Sp5c
		Vehicle	ISDN-3
	Total
	BAL diet	2.85 ± 0.20	3.27 ± 0.08
	TRP -diet	2.84 ± 0.27	3.27 ± 0.14
	V1
	BAL diet	3.23 ± 0.19	3.20 ± 0.23
	TRP -diet	2.91 ± 0.26	3.36 ± 0.12
	V2
	BAL diet	2.85 ± 0.17	3.31 ± 0.14
	TRP -diet	3.13 ± 0.20	3.50 ± 0.18
	V3
	BAL diet	3.02 ± 0.12	3.30 ± 0.17
	TRP -diet	2.94 ± 0.12	2.95 ± 0.18
	Data represent mean ± SEM of (3-5) Sp5C sections from (5-6)
	mice per group. p >0.05 by Tukey's HSD post-hoc test
	following 2-way ANOVA.

Table 3 . Relative mRNA Expression

 3

		Vehicle	ISDN-1	ISDN-3
	[AhR]Sp5c
	BAL diet	108.3 ± 7.0	103.0 ± 14.5	114.7 ± 45.2
	TRP -diet	137.5 ± 11.4	132.0 ± 8.1	74.4 ± 10.1
	[CYP1A1]Sp5c
	BAL diet	100.0 ± 17.3	78.0 ± 12.9	67.2 ± 16.5
	TRP -diet	64.4 ± 13.2	68.1 ± 14.3	58.7 ± 13.9
	[CYP1B1]Sp5c
	BAL diet	100.0 ± 19.7	55.7 ± 8.6	63.4 ± 20.6
	TRP -diet	101.2 ± 25.0	77.7 ± 19.9	68.6 ± 24.0
	[AhRR]Sp5c
	BAL diet	109.0 ± 12.0	142.6 ± 29.0	151.3 ± 23.4
	TRP -diet	149.9 ± 21.6	122.7 ± 10.7	142.5 ± 16.3
	Data represent mean ± SEM in AU of (7-9) mice per group. Samples were
	collected 1 h after ISDN/vehicle administration. p >0.05 by Tukey's HSD post-
	hoc test following 2-way ANOVA.

Table 4 . Summary of Statistical Analyses from 2-way ANOVA Corresponding to Tables 1 to 3 Table F[DFn, DFd] or tDF p-value F[DFn, DFd] p-value Finteraction [DFn, DFd] p-value Table 1

 4 Data represent F values for two-way ANOVA with corresponding degrees of freedom (DFn, DFd), and p-values. ANOVA factors were designed as followed: ISDN, for comparisons of animals injected with ISDN and those with Vehicle; and Diet, for comparisons between BAL and TRP-deficient diets; and Interaction, for the effect between two factors on the dependent variable. p <0.05 was considered statistically significant.

	Total	FDiet[1,18]=9.90	0.006 FISDN[1,18]=2.16 0.16	F[1,18]=0.008	0.93
	V1	FDiet[1,18]=4.89	0.04	FISDN[1,18]=0.15 0.70	F[1,18]=0.12	0.73
	V2	FDiet[1,18]=12.25	0.003 FISDN[1,18]=1.70 0.21	F[1,18]=0.0003	0.98
	V3	FDiet[1,18]=4.95	0.04	FISDN[1,18]=4.35 0.05	F[1,18]=0.0006	0.98
	Table 2
	Total	FDiet[1,17]=8.56x10 -5 0.99	FISDN[1,17]=4.16 0.06	F[1,17]=0.001	0.98
	V1	FDiet[1,17]=0.14	0.72	FISDN[1,17]=0.91 0.35	F[1,17]=1.14	0.30
	V2	FDiet[1,17]=1.69	0.21	FISDN[1,17]=5.30 0.03	F[1,17]=0.05	0.83
	V3	FDiet[1,17]=2.19	0.16	FISDN[1,17]=1.00 0.33	F[1,17]=0.80	0.38
	Table 3
	AhR	FDiet[1,37]=0.12	0.72	FISDN[2,37]=1.07 0.35	F[2,37]=1.87	0.17
	CYP1A1 FDiet[1,35]=2.10	0.16	FISDN [2,35]=0.90 0.42	F[2,35]=0.55	0.58
	CYP1B1 FDiet[1,37]=0.31	0.60	FISDN [2,37]=1.92 0.16	F[2,37]=0.14	0.87
	AHRR	FDiet[1,34]=0.06	0.81	FISDN [2,34]=0.41 0.66	F[2,34]=1.22	0.31

Table 5 . Summary of Statistical Analyses from Figure 1 Table Test F[DFn, DFd] or tDF p-value F[DFn, DFd] p-value Finteraction [DFn, DFd]

 5 Data represent F values for one and two-way ANOVA with corresponding degrees of freedom (DFn, DFd), and pvalues. ANOVA factors were designed as followed: Time, for comparisons at different time points; Diet, for comparisons between BAL and TRP-deficient diets; and Interaction, for the effect between two factors on the dependent variable. p <0.05 was considered statistically significant.

	p-value

ACKNOWLEDGEMENTS

We would like to thank Ms. Karine Herault, Ms. Anne-Marie Gaydier, and Ms.

Sylviane Rousselin for their technical help. All authors declared no conflicts of interest. This work was supported by funding from Institut National de la Santé et de la Recherche Médicale (Inserm), Université Clermont Auvergne (France), CHU Clermont-Ferrand, and Région Auvergne-Rhône-Alpes (CPER DEPICURE2019).

ACKNOWLEDGEMENTS

The authors thank Ms. Tamara Lucien, Ms. Marion Ledevin, and Mr. Rayan Domet for their help with the astrocyte reconstructions; Ms. Manon Meunier for their contribution to behavioral testing; and Ms. Anne-Marie Gaydier and Ms. Sylviane Rousselin for their technical help. All authors reviewed and approved the final manuscript and declared no conflicts of interest. This work was supported by funding from the "Institut National de la Santé et de la Recherche Médicale" (INSERM) and the Clermont Auvergne University (France).

LIST OF TABLES

HYPOTHESIS & OBJECTIVES STUDY I ABSTRACT

Sumatriptan is considered a specific acute treatment for migraine attacks. However, approximately 30% of migraineurs do not respond to it or only partially. The mechanism of this inefficacy is unclear. Tryptophan (TRP), the precursor of 5-HT, has been also involved in migraine pathophysiology. Its acute depletion aggravates ictal episodes and can alter the brain expression of 5-HTR1B. Hence, we aimed to study the contribution of TRP on sumatriptan efficacy in a migraine-like pain model induced by isosorbide dinitrate (ISDN). Two of the most prevalent clinical symptoms (cephalic mechanical hypersensitivity -CMH-and light hypersensitivity -LH-) were evaluated in male and female mice subjected to a TRP-deficient diet for one week. In addition, 5-HT levels and the expression of 5-HTR1B/D were measured. ISDN-induced CMH in both sexes and LH in females only. The TRP-deficient diet reduced plasmatic 5-HT, and significantly prolonged ISDN-induced CMH, but did not worsen the LH in females.

Interestingly, TRP deficiency also blocked the antinociceptive efficacy of sumatriptan on both CMH and LH. It was concomitant with an increased 5-HTR1D expression in the trigeminal nucleus caudalis, without alteration of local 5-HTR1B nor 5-HT levels. Our results suggest that the inability of sumatriptan to block nociception could be associated with an alteration of 5-HTR1D functionality in the trigeminal system, induced by impaired TRP metabolism. It remains to be determined whether these alterations are exclusive to non-responders and whether the action of other triptans is also compromised.

Sumatriptan is no longer efficacious under TRP deficiency

Even with the efficacy shown by sumatriptan, 30-40% of migraineurs report total or partial inefficacy, alone or combined with other analgesic drugs (Viana et al. 2013).

Changes in serotonin levels can be induced through dietary acute depletion of TRP, which is its precursor and is a potent inducer of migraine manifestations including LH and pain (P. D. Drummond 2005).

Quantification of the expression of 5-HTR1B/1D shows a significant increase in the protein expression in the Sp5C, specifically that of 5-HTR1D. Changes solely done at the level of 5-HTR1D could be attributed to its location at the level of primary migrainerelated regions including the LC, hippocampus, and cortex (Tricklebank and Daly 2019). This increase in the expression coupled with the low concentration of 5-HT could contribute to migraine episodes (Panconesi 2008). Moreover, the loss of the efficacy of sumatriptan could be related to receptor functionality as well whereby it is sensitized, rather than expressed. Since sumatriptan acts at the level of the 5-HTR1B/1D, impaired serotonergic neurotransmission, which was reported in migraineurs (Aggarwal, Puri, and Puri 2012), and hence the 5-HTR1B/1D functionality, could impact the triptan efficacy.

Changes in 5-HTR1D expression could be associated with altered functionality, as shown for other 5-HTR1 (Yücel et al. 2016). In this study, we did not examine the sensitivity of receptors to sumatriptan agonism. However, this could be further assessed through the plasma extravasation method which has priorly demonstrated the efficacy of sumatriptan and 5HTR1B in inhibiting the latter following the electrical stimulation of the trigeminal ganglion and hence can be used for 5HTR1D as well (Buzzi and Moskowitz 1990;Moskowitz and Waeber 1996).

CONCLUSION

We showed the pivotal role of TRP metabolism on two of the most prevalent migrainerelated sensory symptoms, the CMH and LH, in mice, as well as on sumatriptan's

SUPPLEMENTARY DATA TRP deficiency does not affect the animal well being

Mice well-being under balanced or TRP-deficient food conditions coupled or not with an ISDN/vehicle administration was evaluated on a daily basis through the measurement of the body weight over 10 days. All mice showed no overall robust change in their weight with similar progression in TRP-deficient and control animals, in both ISDN or vehicle-injected groups (Figures S1A1 andS1A2). In addition, no change was seen in the amount of food consumed during the period of deficiency (Figures S1B1 andS1B2). S1. A-Body weight of female (A1) and male (A2) mice after ISDN or vehicle administration, in balanced (BAL) or TRP-deficient (TRP -) groups. P>0.05 by Tukey HSD posttest following RM 3-way ANOVA. B-Scatter plots showing the food consumption in female (B1) and male (B2) mice in BAL and TRP -groups.

Supplementary Figure

Data represent mean ± SEM of (n) mice per group. p > 0.05 by Student's t-test.

Neither TRP-deficiency nor ISDN induces an anxiety-like behavior

Assessment of anxiety-like behavior was done through measurement of the percentage of time spent in the closed arms during the 5 min of LED-off condition in the mEPM under a balanced and TRP-deficient diet, and/or after an ISDN/vehicle administration (Figure S2). Both male and female mice spent an equal amount of time inside both arms characteristics (including the body and nuclear size) of these neurons were analyzed.

Microglia or astrocyte density and body cell sizes were quantified by IBA1 or GFAP immunolabelling respectively at the level of the TH-IR body cells and projection regions of the LC by using images taken at a 10X magnification.

Morphological and structural analysis of astrocytes

Reconstructions of three-dimensional (3D) GFAP-labeled astrocytes were manually performed from x40 images using the Simple Neurite Tracer plugin of Fiji software.

Cell complexity was analyzed using the fractal dimension approach by the box-counting method obtained from the Fractal Box count tool. Quantifications of the number and the total length of astrocyte branches were performed automatically after 3D reconstruction.

A branch was defined as a continuous projection from the cell body or at a branch point ending. Branch organization around the cell bodies was assessed by using the Sholl analysis tool. Concentric circles around the soma were traced with radii increasing by 2.0 μm. Afterward, the global number of interactions for every astrocyte is added up and graphed as a function of the radial distance from the cell body.

Statistical analysis

The data were presented as mean ± SEM. The number (n) of mice utilized for each analysis was shown in Figures or Tables. 1 andTable S1 from Supplementary Results. The level of significance was set at a p-value below 0.05. All statistical analyses and graphs were performed using GraphPad Prism (v8.4.0, GraphPad Inc., La Jolla, CA, USA). Figures were made using Les objectifs spécifiques de chaque étude ont été les suivants :

SUPPLEMENTARY TABLES

. Étude I :

Évaluer les conséquences d'une déficience alimentaire en TRP sur :

-l'apparition de la douleur de type migraineuse suite à l'administration aigüe de l'ISDN chez les souris males and femelles. Nous avons évalué deux des symptômes sensoriels les plus répandus : l'hypersensibilité mécanique céphalique et l'hypersensibilité à la lumière.

-l'efficacité du sumatriptan (un antimigraineux préconisé pour traiter la crise de céphalée) pour bloquer les hypersensibilités sensorielles induites par l'ISDN.

. Étude II :

-Évaluer la contribution du métabolisme du TRP dans la chronification de la migraine induite par l'administration répétée d'ISDN chez des souris femelles, à travers de l'évaluation comportementale de la sensibilité céphalique, et de la fonction noradrénergique du LC.

-Examiner l'effet de l'administration répétée d'ISDN, seule ou combinée à une déficience en TRP, sur la réponse inflammatoire systémique et locale au niveau du LC.

-Caractériser la contribution des AhR dans la progression de la migraine par l'évaluation comportementale de la sensibilité céphalique après son activation/inhibition pharmacologique, et par l'analyse d'immunofluorescence et biomoléculaire au niveau du LC.

RÉSULTATS . Étude I :

La présente étude a démontré que le déficit chronique du TRP et donc la Quoi qu'il en soit, des recherches plus approfondies sont nécessaires pour comprendre ce domaine.