Dissertations in Health Sciences

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1 PUBLICATIONS OF THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences CINDY GUERRERO TORO THE ROLE OF PURINERGIC, 5-HYDROXYTRYPTAMINERGIC AND GLUTAMATERGIC RECEPTORS IN RAT PERIPHERAL TRIGEMINAL NOCICEPTION: IMPLICATIONS FOR MIGRAINE PAIN

2 The role of purinergic, 5-hydroxytryptaminergic and glutamatergic receptors in rat peripheral trigeminal nociception: Implications for migraine pain

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6 CINDY GUERRERO TORO The role of purinergic, 5-hydroxytryptaminergic and glutamatergic receptors in rat peripheral trigeminal nociception: Implications for migraine pain Peripheral trigeminal nociception in rats To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in Mediteknia Auditorium MD100, Kuopio, on Monday, February 11 th, 2019 at 13:00 Publications of the University of Eastern Finland Dissertations in Health Sciences Number 496 A. I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences University of Eastern Finland Kuopio 2018

7 Grano Oy Kuopio, 2018 Series Editors: Professor Tomi Laitinen, M.D., Ph.D. Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences Professor Hannele Turunen, Ph.D. Department of Nursing Science Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D. Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences Associate Professor (Tenure Track) Tarja Malm, Ph.D. A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences Lecturer Veli-Pekka Ranta, Ph.D. (pharmacy) School of Pharmacy Faculty of Health Sciences Distributor: University of Eastern Finland Kuopio Campus Library P.O. Box 1627 FI Kuopio, Finland h p:// /kirjasto ISBN (print): ISBN (pdf): ISSN (print): ISSN (pdf): ISSN-L:

8 III Author s address: Supervisors: A. I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences University of Eastern Finland P. O. Box 1627, FI KUOPIO FINLAND Professor Rashid Giniatullin, M.D., Ph.D. A. I. Virtanen Institute for Molecular Sciences University of Eastern Finland KUOPIO FINLAND Professor Pasi Tavi, Ph.D. A. I. Virtanen Institute for Molecular Sciences University of Eastern Finland KUOPIO FINLAND Reviewers: Professor An i Pertovaara, M.D., Ph.D. Department of Physiology Faculty of Medicine University of Helsinki HELSINKI FINLAND Professor Michael J. M. Fischer, M.D., Ph.D. Center of Physiology and Pharmacology Medical University of Vienna VIENNA AUSTRIA Opponent: Professor Cristina Tassorelli, M.D., Ph.D. Headache Science Centre - IRCCS C. Mondino Foundation Department of Brain and Behavioral Sciences University of Pavia PAVIA ITALY

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10 V Guerrero Toro, Cindy The role of purinergic, 5-hydroxytryptaminergic and glutamatergic receptors in rat peripheral trigeminal nociception: Implications for migraine pain University of Eastern Finland, Faculty of Health Sciences Publications of the University of Eastern Finland. Dissertations in Health Sciences Number p. ISBN (print): ISBN (pdf): ISSN (print): ISSN (pdf): ISSN-L: ABSTRACT Migraine is a common disabling neurologic disorder characterised by recurrent episodes of headache. Despite the high prevalence, the mechanisms that trigger migraine a acks and the processes leading to the persistent pain in migraine are not completely understood. Nevertheless, a prevailing view suggests that migraine pain results from the activation of trigeminal ganglia neurons and their nerve bres that innervate cranial meninges. In the current study, the role of purinergic, 5-hydroxytryptaminergic and glutamatergic receptors was investigated in rat migraine models. The main focus was on peripheral pain mechanisms generated in the trigeminovascular nociceptive system. Experiments were conducted by using: 1) primary trigeminal ganglia cultures consisting of neurons and satellite glial cells and 2) meningeal preparations containing peripheral trigeminal nerve bres. Control conditions were compared with migraine-like conditions mimicked by the exposure to the migraine mediator calcitonin gene-related peptide (CGRP). In trigeminal neurons, under control conditions, the purinergic, 5-hydroxytryptaminergic, and glutamatergic agonists (i.e. adenosine triphosphate, ATP; 5-Hydroxytryptamine, 5-HT; N-methyl-D-aspartate, NMDA, respectively) were able to evoke calcium responses and variations in the membrane electric potentials; indicating the functional expression of these receptors. Under migraine-like conditions, induced by the exposure to the neuropeptide CGRP, calcium responses were signi cantly elevated. In meninges, as the possible origin site of migraine pain, electrophysiological recordings from trigeminal nerve bres demonstrated that NMDA induced a transient spike ring only in a limited fraction of trigeminal nerve bres. In contrast, a strong and long-lasting nociceptive spiking activity was induced by ATP and 5-HT, suggesting their major contribution to generation of migraine pain. Interestingly, satellite experiments showed that 5-HT, acting via ionotropic 5-HT 3 receptors, triggered peripheral CGRP release in meninges, but inhibited CGRP release in the brainstem; showing the pro-nociceptive peripheral but anti-nociceptive central e ect of 5-HT in migraine models. Taking together, our ndings revealed several novel migraine-pain related mechanisms advancing our knowledge on peripheral trigeminal nociception and suggesting new therapeutical approaches to treat migraine pain at the site of its origin. National Library of Medicine Classi cation: QU 55.7, QU 57, QU 58, QU 60, QV 126, QW F6, QY 60.R6, QY 95, WK 202, WL 102, WL 200, WL 330, WL 342, WL 344, WL 544, WL 704 Medical Subject Headings: Action Potentials; Adenosine; Calcitonin Gene-Related Peptide; Evoked potentials; Glutamates; Headache; Fluorescent Antibody Technique; Ligand-Gated Ion Channels; Meninges; N-Methyl aspartate; Nerve Fibres; Migraine Disorders; Nociception; Nociceptors; Nucleotides; Nucleosides; Pain; Patch- Clamp Techniques; Receptors, G-Protein-Coupled; Receptors, N-Methyl-D-Aspartate; Receptors, Purinergic P2; Receptors, Serotonin; Rats; Sensory Receptor Cells; Serotonin; Trigeminal Nerve.

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12 VII Guerrero Toro, Cindy Purinergisten 5-hydroksi-tryptamiinien ja glutamaa isten reseptoreiden rooli rotan perifeerisessä trigeminaalisessa nosiseptiviassa ja niiden merkitys migreenikivussa Itä-Suomen Yliopisto, Terveystieteiden Tiedekunta Publications of the University of Eastern Finland. Dissertations in Health Sciences Numero s. ISBN (print): ISBN (pdf): ISSN (print): ISSN (pdf): ISSN-L: TIIVISTELMÄ Migreeni on yleinen suorituskykyä heikentävä neurologinen sairaus, jolle luonteenomaisia ovat päänsärkykohtaukset. Huolima a sairauden yleisyydestä migreenikohtauksia laukaisevien tekijöiden ja yhtämi aisen kivun mekanismeja migreenissä ei vielä täysin ymmärretä. Vallitsevan näkemyksen mukaan migreenikipu johtuu trigeminus- eli kolmoishermon ganglion neuroneista ja niiden kova-aivokalvoa hermo avista hermosyistä. Tässä tutkimuksessa purinergisten 5-hydroksitryptamiini- ja glutamaa ireseptoreiden rooli selvite iin rotan kokeellisessa migreenimallissa. Erityisesti keskity iin kipumekanismeihin kolmoishermon hermotusalueen ääreisverenkierrossa ja nosiseptisessa hermotuksessa. Suuri osa kokeista tehtiin: 1) kolmoishermon ganglioviljelmissä, jotka sisälsivät neuroneita, satellii iglia- eli tukisoluja sekä 2) aivokalvoista eriste yjä kolmoishermon hermosyitä. Kokeissa käyte yjä kontrolloituja olosuhteita voidaan verrata migreenin kaltaiseen tilaan, jota mukailtiin altistamalla solut migreeniä laukaisevalle tekijälle, jona käyte iin kalsitoniinigeeniinlii yvää peptidiä (CGRP). Trigeminaalisissa neuroneissa purinergisten 5-hydroksi-tryptamiinien ja glutamaa isten agonistit (i. e. adenosiinitrifosfaa i, ATP; 5-hydroksitryptamiini, 5-HT; N-metyyli-D-aspartaa i, NMDA) saavat aikaan vahvimman kalsiumvasteen ja membraanipotentiaalimuutoksen osoi aen reseptoreiden toiminnallien ilmentymisen. Migreeninkaltaisissa olosuhteissa, jonka aikaansaa altistuminen neuropeptidi CGRP:lle, nämä kalsiumvasteet ovat suurentuneet. Aivokalvoa hermo avista kolmoishermon hermosyistä tehdyt sähköfysiologiset rekisteröinnit osoi ivat, e ä NMDA-reseptorin aktivaatio aikaansai lyhytkestoisen aktiopotentiaalivasteen, joka esiintyi vain pienessä osassa hermosyitä. ATP:n ja 5HT:n anto sen sijaan sai aikaan vahvan ja pitkään kestävän aktiopotentiaalivasteen nosiseptiivissa hermosyissä, mikä vii aa ATP:n ja 5HT:n olevan vahvasti myötävaiku avina tekijöinä migreenikivun synnyssä. Lisäksi kokeet osoi ivat e ä 5-HT aktivoi 5-HT 3 reseptoriväli eisesti CGRP:n vapautumisen aivokalvosta, mu a ei aivorungosta, saaden täten aikaan samanaikaisesti kipua lisäävän ääreishermovasteen ja kipua vähentävän keskushermostovasteen. Yhteenvetona voidaan todeta tutkimuksen paljastaneen monia uudenlaisia migreenikipuun lii yviä mekanismeja. Tulokset laajentavat tietämystä kolmoishermon kipumekanismeista avaten uusia terapeu isia lähestymistapoja hoitaa migreenikipua sen syntypisteessä. Luokitus: QU 55.7, QU 57, QU 58, QU 60, QV 126, QW F6, QY 60.R6, QY 95, WK 202, WL 102, WL 200, WL 330, WL 342, WL 344, WL 544, WL 704 Yleinen Suomalainen asiasanasto: aivot; kipu; migreeni; päänsärky; immunohistokemia; hermosto; hermosolut; nukleotidit; adenosiini; väli äjäaineet; reseptorit; glutamaatit; rotat.

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14 IX Acknowledgements The study presented in this thesis was conducted during the years at the A. I. Virtanen Institute for Molecular Sciences at University of Eastern Finland in Kuopio, Finland. I am deeply grateful to all of those with whom I have had the pleasure to work in the course of this study and other related projects, as well to those who uno cially contribute to review my manuscript. For their supervision and professional guidance, heartfelt thanks to Prof. Dr. Rashid Giniatullin, M.D. Prof. Dr. Pasi Tavi For the examination of this thesis, thanks to Prof. Dr. An i Pertovaara, M.D. Prof. Dr. Michael J. M. Fischer, M.D. For technical support, intellectual input, and academic advice, my sincere thanks to Co-authors: Dr. Abushik, P.; Dr. Fayuk, D.; Dr. Giniatullina, R.; Gubert-Olive, M., M.Sc.; Dr. Ischenko, Y.; Dr. Luz, L. L.; Dr. Kilinç, E.; Dr. Gafurov, O.; Koroleva, K., M.Sc.; Dr. Safronov, B.; Sha gullin, M., M.Sc.; Dr. Shelukhina, I.; Prof. Dr. Sitdikova, G.; Timonina, A., M.Sc.; Prof. Dr. Töre, F., M.D; Vitale, C. M.Sc.; Prof. Dr. Yegutkin, G. G.; Dr. Zakharov, A. Molecular Pain Research group Members of the A. I. Virtanen Institute of Molecular Sciences Members of the Faculty of Health Sciences Professors at University of Eastern Finland Prof. Dr. Malm, T., Editor Dr. Lebrun, J. L., English Editor Mr. Saastamoinen, J., Finnish Editor For their academic and personal support, I profoundly thank to Prof. Dr. Kauppinen, R., M.D. Dr. Andrade, P., M.D. Mr. Cha erjee, B. Family & Friends Cindy Guerrero Toro This study was supported by Finnish Academy; Doctoral program in Molecular Medicine; Faculty of Health Sciences, University of Eastern Finland; Kazan Federal University for the state assignment in the sphere of scienti c activities and the Government of the Russian Federation; Russian Foundation for Basic Research.

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16 XI List of the original publications This dissertation is based on the following original publications: I Yegutkin, G. G., Guerrero-Toro, C., Kilinc, E., Koroleva, K., Ishchenko, Y., Abushik, P., Giniatullina, R., Fayuk, D. & Giniatullin, R. (2016). Nucleotide homeostasis and purinergic nociceptive signaling in rat meninges in migraine-like conditions. Purinergic Signal, 12, II Guerrero-Toro, C., Timonina, A., Gubert-Olive, M. & Giniatullin, R. (2016). Facilitation of serotonin-induced signaling by the migraine mediator CGRP in rat trigeminal neurons. BioNanoScience, 6, III Kilinc, E., Guerrero-Toro, C., Zakharov, A., Vitale, C., Gubert-Olive, M., Koroleva, K., Timonina, A., Luz, L. L., Shelukhina, I., Giniatullina, R., Tore, F., Safronov, B. V. & Giniatullin, R. (2017). Serotonergic mechanisms of trigeminal meningeal nociception: implications for migraine pain. Neuropharmacology, 116, IV Manuscript Guerrero-Toro, C., Koroleva, K., Sha gullin, M., Gafurov, O., Abushik, P., Tavi, P., Sitdikova, G., & Giniatullin, R. The role of glutamate NMDA receptors in peripheral trigeminal nociception implicated in migraine pain. Shared authorship as rst author; these authors contributed equally. The publications were adapted with the permission of the copyright owners. Other original publications by Guerrero-Toro, C. Savchenko, E., Malm, T., Kon inen, H., Hamalainen, R. H., Guerrero-Toro, C., Wojciechowski, S., Giniatullin, R., Koistinaho, J. & Magga, J. (2016). Abeta and In ammatory Stimulus Activate Diverse Signaling Pathways in Monocytic Cells: Implications in Retaining Phagocytosis in Abeta-Laden Environment. Front Cell Neurosci, 10, 279.

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18 XIII Contents ABSTRACT TIIVISTELMÄ ACKNOWLEDGEMENTS LIST OF ORIGINAL PUBLICATIONS CONTENTS ABBREVIATIONS V VII IX XI XIII XVII 1 INTRODUCTION 1 2 TRIGEMINAL NOCICEPTION AND MIGRAINE PATHOPHYSIOLOGY TRIGEMINAL SOMATOSENSORY SYSTEM Peripheral and central a erent pathways of trigeminal nociception Brain regions associated with trigeminal pain MIGRAINE AND TRIGEMINAL NOCICEPTION Epidemiology and diagnosis of migraine Epidemiology and diagnosis of trigeminal neuralgia SIGNALLING PATHWAYS IN THE TRIGEMINOVASCULAR SYSTEM PURINERGIC MECHANISMS IN MIGRAINE ATP as neurotransmi er and neuromodulator ATP as a nucleotide precursor Purinergic receptors Purinergic hypothesis of migraine P2X7 receptors in pain signalling ATP-gated P2X3 and P2X2/3 receptors in pain signalling Purinergic-based drugs for migraine treatment HYDROXYTRYPTAMINERGIC MECHANISMS IN MIGRAINE Hydroxytryptamine Hydroxytryptaminergic receptors Hydroxytryptaminergic hypotheses of migraine Hydroxytryptaminergic-based pharmaceutical drugs for migraine treatment GLUTAMATERGIC MECHANISMS IN MIGRAINE Glutamate as neurotransmi er and neuromodulator in the nociceptive system Glutamatergic receptors Glutamatergic mechanisms of migraine

19 XIV Glutamatergic-based pharmaceutical drugs for migraine treatment CALCITONIN GENE-RELATED PEPTIDE CGRP structure and mechanism of ligand binding Intracellular self-regulatory signalling pathways of CGRP Localisation of CGRP receptors The importance of CGRP for migraine AIMS OF THE STUDY 33 5 MATERIALS AND METHODS ANIMALS CELLS AND TISSUE PREPARATION Isolated hemiskull preparation Trigeminal ganglia cultures Brainstem slices ELECTROPHYSIOLOGY Whole-cell patch-clamp recording Nerve action potentials recording CALCIUM IMAGING HISTOCHEMISTRY Nucleotides staining in hemiskulls Mast cell staining IMMUNOSTAINING FLUORESCENCE-ACTIVATED CELL SORTING (FACS) ENZYMATIC IMMUNOASSAY Determination of purine level Determination of the calcitonin gene-related peptide level STATISTICAL ANALYSIS RESULTS PURINERGIC MECHANISMS IN THE RAT TRIGEMINAL SYSTEM Peripheral trigeminal nerve terminal activation by ATP and its dephosphorylation derivatives in rat meninges The ATP-gated P2X3 receptor expression in trigeminal nerve terminals Calcium responses evoked by ATP and its dephosphorylation derivatives in rat trigeminal ganglia cells The ATP-gated P2X7 receptor expression in satellite glial cells HYDROXYTRYPTAMINERGIC MECHANISMS IN THE RAT TRIGEMINAL SYSTEM Peripheral trigeminal nerve terminals activation by 5-HT in rat meninges The 5-HT receptor mediation of peripheral trigeminal nociceptive responses Calcium responses evoked by 5-HT in rat trigeminal ganglia cells Membrane currents activated by 5-HT in rat trigeminal neurons Cranial meningeal mast cells as 5-HT reservoirs GLUTAMATERGIC MECHANISMS IN THE RAT TRIGEMINAL SYSTEM Peripheral trigeminal nerve terminal activation by NMDA in rat meninges 49

20 XV Calcium responses evoked by glutamatergic agonists in rat trigeminal neurons Membrane currents activated by NMDA in rat trigeminal neurons Glutamate-NMDA receptor subtypes localisation CGRP SENSITISING EFFECT IN RAT MENINGES AND TRIGEMINAL CELLS The e ect of CGRP on extracellular purine homeostasis The e ect of CGRP on nucleotide hydrolysis in rat dura mater The e ect of CGRP on adenine nucleotide responses in trigeminal cells The e ect of CGRP on 5-HT responses in trigeminal cells The e ect of CGRP on NMDA responses in trigeminal cells CGRP release in rat trigeminal cells, the dura mater and the brainstem DISCUSSION PURINERGIC MECHANISMS IN THE RAT TRIGEMINAL SYSTEM HYDROXYTRYPTAMINERGIC MECHANISMS IN THE RAT TRIGEMINAL SYSTEM Targeting peripheral trigeminal 5-HT receptors Targeting central trigeminal 5-HT receptors HT and other receptor interactions in the peripheral trigeminal system Meningeal mast cells as a source of 5-HT GLUTAMATERGIC MECHANISMS IN THE RAT TRIGEMINAL SYSTEM CGRP SENSITISING EFFECT IN RAT MENINGES AND TRIGEMINAL CELLS The e ect of CGRP on purinergic mechanisms in the trigeminal system The e ect of CGRP on 5-hydroxytryptaminergic mechanisms in the trigeminal system The e ect of CGRP on glutamatergic mechanisms in the trigeminal system RESEARCH LIMITATIONS AND CONSIDERATIONS CONCLUSIONS 73 REFERENCES 74 APPENDIX 93 Publication I: Nucleotide homeostasis and purinergic nociceptive signaling in rat meninges in migraine-like conditions Publication II: Facilitation of Serotonin-Induced Signaling by the Migraine Mediator CGRP in Rat Trigeminal Neurons Publication III: Serotonergic mechanisms of trigeminal meningeal nociception: Implications for migraine pain Publication IV: The role of glutamate NMDA receptors in peripheral trigeminal nociception implicated in migraine pain List of compounds

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22 XVII Abbreviations 5-HT 5-Hydroxytryptamine GPCR G protein-coupled receptor ACSF Arti cial cerebrospinal uid iglur Ionotropic glutamate receptor Ado Adenosine KCl Potassium chloride ADP Adenosine diphosphate LIC Ligand-gated ion channels AMP Adenosine monophosphate mglur Metabotropic glutamate receptor AMPA ATP α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid Adenosine triphosphate NMDA N-methyl-D-aspartate P2X Purinergic ligand-gated ion receptor BSA BSS Ca 2+ camp CGRP CLR Bovine serum albumin Basic salt solution Calcium (ion) Cyclic adenosine monophosphate Calcitonin gene-related peptide Calcitonin receptor-like receptor P2Y PKA PKC PNS RAMP Purinergic G-protein-coupled receptor Protein kinase A Protein kinase C Peripheral nervous system Receptor activity-modifying protein CNS Central nervous system SGC Satellite glial cell CREB CSD CSF camp response element-binding protein Cortical spreading depression Cerebrospinal uid SpVc TNC TRPV1 Spinal trigeminal nucleus Trigeminal nucleus caudalis Transient receptor potential cation channel subfamily V member 1 ERK Extracellular signal regulated kinase VPM Ventral posteromedial nucleus GABA Gamma-aminobutyric acid

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24 1 Introduction Someone has divided headaches into three simple groups, viz., those that one can forget, those that one cannot forget, and those that make one forget everything else. The headache of migraine falls in the third group. Sco (1941). Migraine is one of the oldest ailments known to mankind. Despite this fact, its pathophysiological mechanism remains largely unknown. During the Hippocratic period, there was no clear di erentiation between the types of headache, and therefore a non-description per se of migraine. During the second century A.D., Aretaeus from Cappadocia brought the term heterokranie to describe a half-sided headache with intermi ent pain manifestations. Later, Galen said: In the healthy state, there would be connections between the vessels within and outside the skull, through which the excess vapour or liquids could escape. Whether this connection is hindered, some body parts would send the brain liquids or vapours of the bad kind. indicating that any disturbance in the cranial vasculature may have detrimental health consequences. Furthermore, Caelius Aurelianus ( fourth century A.D.), described that headache could also be accompanied by visual disturbances (Flatau, 1912). Nowadays, one can still nd resemblances between the heterokranie from Aretaeus and hemiplegia on migraine; the description by Galen and the vascular hypothesis of migraine on cranial meninges; or the headache symptoms described by Caelius Aurelianus and the neurological manifestations during migraine with aura. All these well-known and well documented reports through history to our days, transmit how important migraine is as a neurological disorder. There is a wide misconception between headache and migraine within the average global population. Clarifying, migraine is a type of headache, but not every headache is migraine. Headache is one of the most frequent medical complaints, and it has a high incidence (46%) among the general adult population (Leonardi et al., 1998; Stovner et al., 2007). The di erential diagnosis of the di erent types, subtypes, and subforms relies on their cause, recurrence, localisation, and other symptoms. The International Headache Society (IHS) classi ed hierarchically the types of headaches (see Table 1) into three main groups: Table 1. Headache classification. Type of headache ICHD * Diagnosis Primary headache Secondary headache Painful cranial neuropathies, other facial pains and other headaches Migraine Tension-type headache Trigeminal autonomic cephalalgias Other primary headache disorders Headache a ributed to trauma or injury to the head and/or neck Headache a ributed to cranial or cervical vascular disorder Headache a ributed to non-vascular intracranial disorder Headache a ributed to a substance or its withdrawal Headache a ributed to infection Headache a ributed to disorder of homoeostasis Headache or facial pain a ributed to disorder of the cranium, neck, eyes, ears, nose, sinuses, teeth, mouth or other facial or cervical structure Headache a ributed to psychiatric disorder Painful cranial neuropathies and other facial pains Other headache disorders * ICHD, Code for International Classi cation of Headache Disorders, 3rd edition. Adapted from Headache Classi cation Commi ee of the International Headache (2013).

25 2 1) Primary headaches based on headache symptoms as the major pathology. 2) Secondary headaches based on the causes of headache and whether headache symptoms are secondary to any other disorder. 3) Painful cranial neuropathies, other facial pains, and other headaches. Migraine is the most common type of headache (see Fig. 1) and it is in the top ten most prevalent neurological disorders (Vos et al., 2016). Migraine is a primary headache. However, it can be considered a secondary headache only if it is a symptom of any another disorder (symptomatic migraine). Headache can result from the activation and sensitisation of the trigeminovascular system, which includes pain-sensitive structures associated to dura mater and its vasculature, cervical prolongations of the trigeminocervical complex (TCC), and central projections to the trigeminal nucleus caudalis (TNC) and their nerve terminals (Bartsch and Goadsby, 2003; Shimizu and Suzuki, 2010). The trigeminal nerve plays a very important role in headache nociception (perception or sensation of pain; see section 2.1.1). The primary a erent neurons transmit nociceptive signals from pain-sensitive terminals located in cranial structures through the trigeminal ganglia to the brainstem. Then, this information is relayed to the central nociceptive pathways in the spinal trigeminal nucleus which discriminate pain and temperature (Shimizu and Suzuki, 2010). The major primary pathologies associated to trigeminal nerve activation and sensitisation are migraine and trigeminal neuropathy. Migraine is the most prevalent of these. This disabling chronic neurological disorder (Menken et al., 2000) desperately needs new therapeutical approaches based on new insights of its pathophysiology, that would improve quality of life of these patients and minimise social burden underlying this disorder. Scholarly Output, unit The role of 5-HT on migraine (Sicuteri, 1959) The role of purines on migraine (Burnstock, 1989) CGRP release during activation of the trigeminovascular system (Goadsby et al., 1988) Glutamate on migraine (Hypothesis) (Pollack and French, 1975) Headache Migraine Publication year Figure 1. PubMed trend benchmark for headache and migraine. Timeline showing the increasing number of publications for "headache" (orange) and "migraine" (blue) in PubMed during the last century. Headache is the most striking symptom in diverse disorders, such as: tension-type headaches, cluster headaches, and migraine. Leading articles involving purines (Burnstock, 1989), 5-hydroxytryptamine (Sicuteri, 1959), glutamate (Pollack and French, 1975), and calcitonin gene-related peptide (CGRP; Okragly et al., 2017) on migraine pathophysiology.

26 2 Trigeminal nociception and migraine pathophysiology 2.1 TRIGEMINAL SOMATOSENSORY SYSTEM The pain in the head and neck is mediated by various nerve bres in the nervus intermedius (part of facial nerve) and the trigeminal, glossopharyngeal, and vagus nerves. The trigeminal nerve (cranial nerve V) and its prolongations into the head is one of the twelve cranial nerves responsible for the transmission of stimuli involving tactile, proprioceptive, as well as, pain and temperature sensation. The trigeminal nociceptive signalling starts from the nerve terminals widely distributed on the head, including tissues such as meninges covering the brain. This nociceptive (pain) signalling is guided to reach the brainstem, thalamus, cerebral cortex, cerebellum and reticular formation (Joo et al., 2014) and could nally be perceived as headache Peripheral and central a erent pathways of trigeminal nociception The current knowledge suggests that the initiation of the headache involves the activation of primary a erent neurons innervating cranial structures, such as the meninges (including pia, arachnoid, and dura mater), meningeal vasculature, and the cranial and cervical muscles and connective tissue. These sensory neurons (nociceptors) are located in the trigeminal and dorsal root ganglia of the upper cervical spinal nerves (Noseda and Burstein, 2013; Shimizu and Suzuki, 2010). The nociceptors can be activated by mechanical, electrical or chemical stimulation to transmit nociceptive signals to higher pain centres via nerve bres. Meninges and intracranial vessels are innervated by small-calibre, unmyelinated, slow-conducting bres (C- bres) and small-diameter, thinly myelinated, more rapid-conducting bres (Aδ bre; Liu et al., 2008). Studies suggested that C- bres transmit aching, throbbing or burning sensation, while Aδ bres conduct a sharp pain sensation (Shimizu and Suzuki, 2010). The primary a erent bres emerge from neurons located in the trigeminal ganglion that gives origin to the ophthalmic (V1), maxillary (V2), and mandibular (V3) divisions of the trigeminal nerve. The trigeminal system has three sensory nuclei linked with trigeminal a erents which form a column of cells from mid-brain to the upper cervical spinal cord (see Table 2). Table 2. Components of the trigeminal somatosensory ascending pathway. Primary a erents (cell bodies) Primary a erents (central processes) Second order neurons Ascending pathway Destination Touch & proprioception Trigeminal ganglion, mesencephalic nucleus Entering trigeminal bres Main sensory nucleus Medial lemniscus, posterior trigeminothalamic VPM * Pain & temperature Trigeminal ganglion Spinal tract Spinal nucleus (caudal nucleus) Spinothalamic tract VPM and others * VPM, ventral posterolateral nucleus. Adapted from Vanderah and Gould (2016). The rst nucleus, the main sensory nucleus (Pr5, principal trigeminal nucleus), discriminate tactile and proprioceptive sensations (see Fig. 2 A). Pr5 is located next to the motor nucleus, in the mid-pons, lateral to the middle cerebellar peduncle. The nerve bres from Pr5 have two ascending pathways that reach the ventral posteromedial (VPM) nucleus of thalamus: anterior trigeminothalamic tract (contralateral) and posterior trigeminothalamic tract (ipsilateral).

27 4 The posterior trigeminothalamic tract does not join the medial lemniscus and end in its own independent region of VPM. A Postcentral gyrus VPL VPM B Postcentral gyrus VPL, VPM, other nuclei Medical lemniscus Posterior trigeminal tract Medical lemniscus Spinothalamic tract Medial lemniscus Rostral midbrain Cerebral peduncle Trigeminal ganglion Rostral midbrain Cerebral peduncle Trigeminal ganglion Midpons Caudal medulla From touch, proprioceptors Main sensory nucleus Spinothalamic tract Midpons Caudal medulla From pain, temperature, and some touch and pressure receptors Spinal tract VII, IX, X Spinal nucleus V3 V2 V1 C8 From touch, proprioceptors C8 From pain, temperature, and some touch and pressure receptors L4 L4 Figure 2. The trigeminal somatosensory pathways. Schematic representation of ascending trigeminal pathways A. from the main sensory nucleus and B. from the spinal nucleus. The inset located next to caudal medulla details the organisation of fibres within the spinal trigeminal tract of the cranial nerve V (trigeminal) and its three subdivisions (V1, ophthalmic; V2, maxillary; V3, mandibular) and cranial nerves VII (facial), IX (glossopharyngeal), and X (vagus) that innervate regions of the head and neck. Abbreviations: C8 cervical spinal disc 8; L4, lumbar spinal disc 4; VPL, ventral posterolateral nucleus; VPM, ventral posteromedial nucleus. Adapted from "Cranial nerves and their nuclei" (p. 315, 317) by Vanderah and Gould. In: "Nolte's The Human Brain. An Introduction to its Functional Anatomy", 2016, Philadelphia, PA: Elsevier. Copyright 2016 by Elsevier, Inc. The second nucleus, the spinal trigeminal nucleus (SpVc or Sp5), discriminates pain and temperature (see Fig. 2 B). Primary sensory a erents bres enter the pons (located in the brainstem, via spinal trigeminal tract) passing caudally while leaving collaterals that extend and reach the SpVc and upper cervical spinal cord (C1-C3). The C- and Aδ nociceptive a erent bres lead predominantly to laminae I and II and some Aδ bres go to the lamina V of SpVc (Liu et al., 2008; Noseda and Burstein, 2013). The spinal trigeminal tract extends near to the third cervical segment, allowing the SpVc to progressively fuse with the posterior horn and the spinal trigeminal tract gradually blends with the posterolateral tract (Noseda and Burstein, 2013; Vanderah and Gould, 2016). The SpVc has three subnuclei, named according to their position relative to the obex: caudal nucleus, oral nucleus, and interpolar nucleus. Interestingly, there is also an alternative ascending pathway for small-diameter trigeminal a erents involved in pain and temperature (see Fig. 2 B). These a erents descend at midpons level, via the spinal trigeminal tract, and synapse with second-order neurons in the caudal nucleus at the caudal medulla level. Posteriorly, the trigeminal pain information is driven to

28 5 the thalamus by relays in the reticular formation. The third nucleus, the mesencephalic nucleus (Me5, extends rostrally), which is involved in proprioception. The information from primary a erents is driven to the trigeminal motor nucleus that is associated with proprioception for mastication. It innervates muscles of the rst pharyngeal arch (masseter, temporalis, medial and lateral pterygoid; Vanderah and Gould, 2016) Brain regions associated with trigeminal pain The trigeminal neuronal projections reach di erent regions of the thalamus, such as the VPM, posterior (Po) and lateral posterior/dorsal (LP/LD) nuclei (see Fig. 3). The neuronal corti- A S1 S2 AI PtA RSA Au V1/V2 Ect Rat M1/M2 Cortex 3V LD/LP Po VPM PF Thalamus Hypothalamus LPO AH LH PH Midbrain PB 4V ctg Pons Cerebellum Medulla Oblonga SpVc C1/C2 Scalp Bone Dura Arachnoid Pia Superior sagi al sinus Cortex Trigeminal Ganglion B M1/M2 S1 PtA Cortex Ins S2 Scalp Bone Dura Arachnoid Pia Human Superior sagi al sinus Cortex Hypothalamus Lp/Pul Po VPM Au RS Ect Thalamus Cerebellum PAG PB SSN Trigeminal Ganglion Figure 3. Schematic representation of the ascending neuronal pathways of the trigeminovascular system from the meninges to the cortex A. in rat and B. human. The peripheral limb contains nociceptors that communicate from meningeal terminals to the spinal trigeminal nucleus (SpV and C1/C2, in blue). The ascending limb from SpVc to brainstem, thalamus, and the hypothalamus (in red). Thalamocortical projections from VPM, Po, LD/LP nuclei (in purple, green and ochre respectively). Abbreviations: AH, anterior hypothalamus; AI, agranular insular cortex; ctg, central tegmental area; Ect, ectorhinal cortex; Ins, insula; Au, auditory cortex; LH, lateral hypothalamus; LP/LD, lateral posterior/dorsal nucleus; LPO, lateral posterior area; M1/M2, primary/secondary motor cortex; PAG, periaqueductal grey; PB, parabrachial area; Pf, parafascicular thalamus; PH, posterior hypothalamus; PtA, parietal association cortex; Po, posterior nucleus; RS, retrosplenial cortex; S1/S2, primary/secondary somatosensory cortex; SSN, superior salivatory nucleus; V1/V2, primary/secondary visual cortex; VPM, ventral posteromedial nucleus. Adapted from (A) Noseda and Burstein, 2013, Pain, 154, p Copyright 2010 by Elsevier B.V. (B) Noseda et al., 2011, J Neurosci, 31, p Copyright 2011 by the authors. SpVc V1/V2

29 6 cal projections of the trigeminal system are determined by their origin in the thalamic nuclei (Noseda and Burstein, 2013). In general, neurons that originate from the VPM nucleus project to trigeminal primary (S1) and secondary (S2) somatosensory, and insular cortex have implications on the perception of headache. The Po and LP/LD nuclei neurons project to nontrigeminal areas of S1 (auditory, visual, retrosplenial, and ectorhinal regions), which may be involved in secondary headache pain experience, like the impairment or functional decrease in motor, vision, auditory, memory, and cognitive performance (Noseda et al., 2011). Functional abnormalities in the trigeminal system or the activation of trigeminal primary a erent neurons by an external noxious stimulus may trigger diverse pathological conditions associated with pain, such as trigeminal neuralgia and migraine. 2.2 MIGRAINE AND TRIGEMINAL NOCICEPTION Epidemiology and diagnosis of migraine Migraine is a common chronic disabling primary neurologic disorder characterised by a prominent throbbing headache. It is generally associated with light, sound, and/or smell hypersensitivity, as well as multifarious autonomic (including nausea) and neurological (motor and cognitive) symptoms and emotional disturbances (Noseda and Burstein, 2013). Migraine has a high socio-economic impact. The Global Burden of Disease Study 2015 indicated that migraine is the third-highest cause of disability in middle age population (25-50 years old) and the seventh most prevalent disorder worldwide (Vos et al., 2016). The incidence of this disorder is found to be more prevalent in women than men (3:1 female-to-male ratio; Burstein et al., 2015; Pietrobon and Striessnig, 2003). Migraineurs often present characteristic warning signals (prodrome phase) occurring 24 to 48 hours prior to the onset of headache. The prodrome phase can include increased fatigue, neck sti ness, depression, yawing, euphoria, irritability, constipation, and/or abnormal hypersensitivity to light, sound, and/or smell (Burstein et al., 2015). According to the Headache Classi cation Commi ee of the International Headache (2013), migraine consists predominantly of two major types: migraine without aura, which is a clinical syndrome identi ed by a headache with characteristic symptoms (see Table 3) and migraine with aura, which can be identi ed by the transient focal neurological symptomatology generally accompanied or preceded by headache. Once throbbing of the headache ceases a postdrome phase starts, in which migraineurs feel themselves exhausted, dizzy, and weak or with decreased energy. In addition, a chronic migraine condition is diagnosed if a headache occurs 15 or more days per month (with minimum 8 days with migraine headache symptoms) for more than 3 months. Ascribable to the wide variation in clinical manifestations on migraine with aura, the Headache Classi cation Commi ee of the International Headache (2013) assigned a more re ned classi cation (see Fig. 4), in which, the main subtypes are: migraine with typical aura, accompanied or not by headache (within 60 minutes); migraine with brainstem aura, originated from brainstem but no motor weakness; hemiplegic migraine, characterised by motor weakness (it may occur either in family members or in one individual); and retinal migraine, characterised by repeated reversible a acks in ocular area (monocular scotomata or blindness) Epidemiology and diagnosis of trigeminal neuralgia The trigeminal neuralgia (TN) is a very painful neuropathy that involves directly the trigeminal nerve. The classical trigeminal neuralgia is characterised by recurrent unilateral sharp electric shock-like pain that compromises one or more branches of the trigeminal nerve (ophthalmic, maxillary, and mandibular). It may be triggered without apparent cause or by innocuous stimuli (De Toledo et al., 2016; Headache Classi cation Commi ee of the International Headache, 2013).

30 7 Table 3. Diagnosis criteria for migraine. Migraine without aura Migraine with aura A. No less than ve a acks ful lling criteria B D A. No less than two a acks ful lling criteria B and C B. Headache a acks last 4 to 72 hours (untreated or unsuccessfully treated) C. Headache present with at least two characteristics: 1. unilateral location 2. pulsating quality 3. moderate to severe pain 4. Aggravated by routine physical activity D. Headache present at least one characteristic: 1. Nausea, vomiting or both 2. Photophobia and phonophobia B. Present one or more temporary characteristic aura symptoms: 1. Visual 2. Sensory 3. Speech and/or language disturbances 4. Motor 5. Brainstem 6. Retinal C. Presenting at least two of the underneath characteristics: 1. At least one aura symptom progressively propagates for 5 minutes, ensuing two or more symptoms 2. Each aura symptom endures 5 to 60 minutes 3. At least one aura symptom should be unilateral 4. The aura phase is accompanied, or followed in under 60 min, by headache D. Not be er accounted diagnosis by another ICHD3b * condition. E. Not be er accounted diagnosis by another ICHD3b * condition * ICHD3b, International Classi cation of Headache Disorders, 3rd edition (beta version). Adapted from Headache Classi cation Commi ee of the International Headache (2013). Migraine Migraine without aura Migraine with aura Chronic migraine Migraine with typical aura Migraine with brainstem aura Hemiplegic migraine Retinal migraine Typical aura with headache Typical aura without headache Familial hemiplegic migraine (FHM) Sporadic hemiplegic migraine FHM type I FHM type II FHM type III FHM other loci Figure 4. Classification of migraine. Migraine is constituted by two main subtypes, migraine with aura and migraine without aura. Chronic migraine is considered if a headache occurs on 15 or more days per month during a period superior to three months with migraine symptoms (see Table 3). The familial hemiplegic migraine (FHM) type I has a causative mutation in gene CACNA1A, FHM type II in gene ATP1A2, FHM type III in gene SCN1A, and FHM other loci it is determined when a genetic test shows no mutation on CACNA1A, ATP1A2 or SCN1A genes. Trigeminal neuralgia is an uncommon neuropathy with a higher incidence in women than men (3:1 female-to-male ratio; De Toledo et al., 2016). The lifetime prevalence of TN is 0.3% with an annual incidence of 4.3 to 27 per inhabitants (Lin et al., 2015). Regardless of the low incidence of TN, this is the most common neuralgia in elders, the incidence increases with age (De Toledo et al., 2016). Trigeminal neuralgia is caused by compression of one or more branches of the trigeminal

31 8 nerve. The maxillary and mandibular branches are predominantly a ected by this pathology. A neurovascular compression can incur in a classical trigeminal neuralgia, particularly by pressure increased in the superior cerebellar artery, although an aberrant loop of an artery or vein seems to be the source in most of the cases. Classical trigeminal neuralgia most frequently a ects the second and third branches of the trigeminal nerve ipsilateral (same side) to stimuli, but never contralateral (opposite side), however it can occur bilateral. Individuals can manifest mild autonomic symptoms like epiphoral and/or redness of the eye. During severe pain a acks, the muscles of the face could be a ected ipsilateral to initial stimuli. The individuals a ected are asymptomatic between paroxysms (sudden manifestation of symptoms). During symptomatic phases, the pain may vary in time and intensity, but typically an individual with more frequent a acks tends to have more pain within paroxysms. In the case of classical trigeminal neuralgia with concomitant facial pain during a acks, the a ected area remains with background facial soreness after the a ack (Headache Classi cation Commi ee of the International Headache, 2013; Love and Coakham, 2001). Despite the number of trigeminal neuropathies, the diagnosis of TN depends on the medical history of the patient. The international Headache Society a ributed a diagnosis of TN when characteristic symptoms (see Table 4) are not caused by herpes zoster infection, tumour, multiple sclerosis, or trauma. In addition, increased evidence shows that the activation of the peripheral trigeminovascular system not only plays a role in TN but also in migraine pathophysiology (Burstein et al., 1998; Olesen et al., 2009), because both can involve trigeminal nerve pathways and blood vessels. However, the underlying mechanism that links both pathologies requires further studies. Table 4. Diagnosis criteria for trigeminal neuralgia. Classical trigeminal neuralgia A. No less than three a acks of unilateral facial pain ful lling criteria B D B. Occurring in one or more branches of the trigeminal nerve, with no radiation beyond the trigeminal distribution C. Pain present at least three characteristics: 1. Recurring in paroxysmal a acks (lasting s) 2. Severe intensity 3. Electric shock-like, shooting, stabbing or sharp in quality 4. Triggered by innocuous stimuli D. No evidence of clinical neurological de cit E. Not be er accounted diagnosis by another ICHD3b * condition Classical trigeminal neuralgia, purely paroxysmal A. Recurrent a acks of unilateral facial pain ful lling criteria for classical trigeminal neuralgia B. No persistent facial pain between a acks C. Not be er accounted diagnosis by another ICHD3b * condition Classical trigeminal neuralgia with concomitant persistent facial pain A. Recurrent a acks of unilateral facial pain ful lling criteria for classical trigeminal neuralgia B. Persistent facial pain of moderate intensity in the a ected area C. Not be er accounted diagnosis by another ICHD3b * condition * ICHD3b, International Classi cation of Headache Disorders, 3rd edition (beta version). Adapted from Headache Classi cation Commi ee of the International Headache (2013).

32 3 Signalling pathways in the trigeminovascular system 3.1 PURINERGIC MECHANISMS IN MIGRAINE ATP as neurotransmi er and neuromodulator ATP (adenosine 5 -triphosphate) is best known as the major energetic nucleotide predominantly produced in the mitochondria by oxidative phosphorylation (Bodin and Burnstock, 2001; Brand and Nicholls, 2011). ATP can be released from di erent types of cells, such as astrocytes, neurons, endothelial cells, mast cells, platelets, urothelial cells, osteoblasts, and broblasts (Burnstock, 2016b; Burnstock et al., 2011; Burnstock and Ralevic, 2014; Fabbro et al., 2004; Pangrsic et al., 2007). Intracellular ATP could be released into extracellular space as a consequence of di erent physiological (Bodin and Burnstock, 2001) and pathological conditions, predominantly by the mechanical response to shear stress, stretch, osmotic di erences, hypoxia, and external agents (Burnstock, 2016b, 2017a). The main source of extracellular ATP is programmed cell death, succeeded by in ammatory cell responses, such as endothelial cells during acute in ammation (Burnstock, 2016a). However, ATP is not only released under pathological pro-apoptotic processes (Lazarowski, 2012), but also during healthy physiological conditions (Bodin and Burnstock, 2001). Extracellular ATP also plays a role in the regulation of di erent biological processes, like cardiac function, muscle contraction, vasodilatation, liver glycogen metabolism, in ammation, neurotransmission, and neuroprotection (Burnstock, 2006; Burnstock and Knight, 2004). In addition, ATP is also known to be a co-transmi er in the sympathetic nerves that supply both visceral and cardiovascular systems (Burnstock, 2004), as well as, in parasympathetic nerves that supply the urinary bladder (Burnstock, 2006), making extracellular ATP an important signalling molecule to modulate many vital functions of the body and a potential injurious agent if not regulated otherwise ATP as a nucleotide precursor ATP can be dephosphorylated to produce other adenosine nucleotides (see Fig. 5), like adenosine diphosphate (ADP) and adenosine monophosphate (AMP). Conversion of ATP into ADP and AMP is mainly controlled by the nucleoside triphosphate diphosphohydrolase (NTPDase) enzyme family: ecto-nucleoside triphosphate diphosphohydrolase (e-ntdpase) and ectonucleotide pyrophosphatase/phosphodiesterase (e-npp). Adenosine is a biologically active nucleoside primarily produced by the ecto-enzymatic breakdown of AMP by ecto-5-nucleotidase enzyme (also known as CD73; Yegutkin et al., 2016; Zimmermann et al., 2012). Analogous to ATP, adenosine can be released directly by certain subpopulations of neurons and glial cells (Wall and Dale, 2007). Adenosine is also a powerful homeostatic- and neuromodulator in the central nervous system, acting via A1 receptor at the synaptic level. The ability of adenosine to diminish neuronal damage by inhibiting of the excitatory transmission suggests a fundamental role of this compound in neuroprotection (Cunha, 2001). Variations in adenosine concentration may have a dramatic functional implication on the intracellular equilibrium of purines, therefore, adenosine activity is highly regulated by the conversion into inactive inosine or by a re-uptake pathway Purinergic receptors The role of ATP acting as an extracellular signalling molecule was originally proposed by Burnstock (1972). According to his view, ATP acts as a functionally active co-transmi er in peripheral and central nerve bres (Burnstock, 2007a). Initially, two types of purinergic re-

33 10 ATP UTP - NTPDase1, -2, -3, -8 - alkaline phosphatase ecto-nucleoside diphosphokinase - NTPDase4, -8 - alkaline phosphatase NPP1, -2, -3 ADP - NTPDase1, -3 - NPP1, -2 - alkaline phosphatase AMP kinase UDP IMP AMP deaminase - ecto-5'-nucleotidase - NPP2 (autotaxin) - alkaline phosphatase AMP adenosine adenosine kinase APRT PNP NPP1, -2, -3 adenine camp adenosine deaminase ecto-5'-nucleotidase inosine uric acid Figure 5. Conversion pathways of ATP. Dephosphorisation of ATP is a favourable process to produce adenosine, inosine, and energy. This process involves several enzymatic catalytic steps by specific kinases, which additionally generate secondary products such as: IMP, UTP/UDP, adenine, camp, and uric acid. Abbreviations: APRT, adenine phosphoribosyltransferase; camp, cyclic adenosine monophosphate; IMP, inosine monophosphate; PNP, purine nucleoside phosphorylase; UDP, uridine diphosphate; UTP, uridine triphosphate. Adapted from "Adenosine 5'-triphosphate and adenosine as endogenous signalling molecules in immunity and inflammation" by Bours et al., 2006, Pharmacol Ther, 112, p Copyright 2006 by Elsevier, Inc. ceptors were de ned: the purinoceptor P1 (sensitive to adenosine) and P2 (activated by ATP and/or ADP). Later, the subclasses of P1 and P2 receptors were proposed as P2X (ligand-gated ion channels, LIC; also known as ionotropic) and P2Y (G protein-coupled receptor, GPCR; also known as metabotropic) receptors (see Table 5; Burnstock, 1972, 2007b). Some of the P2Y and P2X receptors subtypes have a predominant role in pain transmission (see Table 6). Furthermore, Burnstock (1981) also proposed that extracellular ATP had a role in migraine pathology. According to his purinergic hypothesis of migraine cerebral blood ow decreases during a pre-headache phase of migraine while blood ow increases during the headache phase and ATP, along with its breakdown products, such as AMP and adenosine, mediate the vasodilatation. Table 5. Properties of purinergic receptors. Receptor Main distribution Main function Agonist Antagonist P1 A1 Brain, spinal cord, testes, heart, and autonomic nerve terminals 1. Prejunctional neuromodulation of neurotransmi er release 2. Behavioural e ects (sedation, anticonvulsive, and anxiolytic) 3. Cardiac depression 4. Non-vasoconstrictor anti-nociceptive e ect CPA CHA R-PIA ADAC NECA 2-Cl-Ado Adenosine S-PIA * DPMA * CPX XAC CPT 8-PT CGS Theophylline * 8-pSPT * IBMX * KF *

34 11 A2A Brain, heart, lungs, and spleen 1. Facilitates neurotransmission 2. Smooth muscle relaxation A2B Large intestine and bladder 1. Role in allergic and in ammatory disorders 2. Vasodilatation A3 Lung, liver, brain, testes, and heart 1. Facilitates release of allergic mediators 2. Cardioprotective and cytoprotective Ligand-gated ion P2 P2X1 P2X2 P2X3 Smooth muscle, platelets, cerebellum, and dorsal horn spinal neurons Smooth muscle, CNS, retina, chroma n cells, autonomic and sensory ganglia, thalamus, and hypothalamus Sensory neurons, NTS, and some sympathetic neurons 1. Smooth muscle contraction (ligand ATP) 2. Platelet activation Sensory transmission and modulation of synaptic function 1. Mediates sensory transmission 2. Facilitates glutamate release in CNS 3. Long-lasting nociception P2X4 CNS, testes, and colon Modulates chronic in ammatory and neuropathic pain P2X5 Proliferating cells in skin, gut, bladder, thymus, spinal cord, proprioceptive neurons of mesencephalic nucleus (Me5), and sensory ganglia Inhibits proliferation and increase di erentiation NECA CGS APEC Adenosine 2-Cl-Ado CV 1808 R-PIA ADAC CPA * CHA * S-PIA * NECA 2-Cl-Ado Adenosine R-PIA S-PIA * APNEA N-benzyl- NECA NECA R-PIA CGS * ATP MeSATP α,β-meatp ATP-γ-S PAPET-ATP β,γ-cf 2 ATP ATP MeSATP ATP-γ-S AP 4A ATP MeSATP α,β-meatp ATP-γ-S Ivermectin ATP MeSATP ATP-γ-S GTP ATP MeSATP α,β-meatp ATP-γ-S XAC CSC KF CGS CPT CPX 8-PT DMPX * 8-pSPT * IBMX * Theophylline * XAC CPX 8-PT CGS pSPT Theophylline DMPX * IBMX * BW-A522 Aromatic sulfonates NF449 NF279 PBS-1011 * TNP-ATP Ip 5I isoppads TNP-ATP Suramin Dihydroxypyridine nicardipine NF279 NF770 NF778 PBS-1011 RB2 Ip 5I A TNP-ATP Suramin PBS-1011 * A RO3 RO4 PBS-1011 TNP-ATP RB2 BBG PSB BBG PPADS Suramin TNP-ATP

35 12 P2X6 CNS, motor neurons in spinal cord, and sensory ganglia Functions as a heteromeric channel in combination with P2X2 and P2X4 subunits ATP MeSATP α,β-meatp ATP-γ-S TNP-ATP Suramin P2X7 Apoptotic cells (e.g., immune cells, pancreas, skin), glial cells, and macrophages 1. Mediates apoptosis, cell proliferation and proin ammatory cytokine release 2. Maintenance of nociception sensitivity (neural-glial cell interactions) ATP Bz-ATP A A NF279 PBS-1011 AZD9056 KN-62 BBG G-protein coupled P2 P2Y1 Epithelial and endothelial cells, platelets, immune cells, osteoclasts, glial cells, and brain 1. Smooth muscle relaxation and mitogenic actions 2. Platelet shape change and aggregation 3. Bone resorption ADP 2-MeSADP ADP-β-S MRS2365 MRS2500 MRS2179 P2Y2 Immune cells, epithelial and endothelial cells, kidney tubules, osteoblasts, and astrocytes 1. Vasodilatation via endothelium and vasoconstriction via smooth muscle 2. Mitogenic actions 3. Mediates surfactant secretion 4. Epithelial cell Cl secretion 5. Bone remodelling 6. Inhibits plasma protein extravasation UTP/ATP MRS2698 MRS2768 UTP-γ-S INS37217 Ap 4A PSB-716 AR-C Suramin RB2 P2Y4 Endothelial and endothelial cells, intestine, pituitary, brain, placenta, spleen, thymus, low levels in liver, and bone marrow 1. Regulates epithelial chloride transport 2. Vasodilatation via endothelium 3. Mitogenic actions UTP/ATP ATP-γ-S 2 -Azido-2 - deoxyutp ATP (for human) RB2 Suramin P2Y6 Airway and intestinal epithelial cells, placenta, T cells, thymus, spleen, kidney, and activated microglia 1. NaCl secretion in colonic epithelium 2. Role in epithelial proliferation UDP PSB-0474 UDP-β-S INS48823 MRS2693 MRS2578 P2Y11 Spleen, intestine, brain, and granulocytes 1. Role in maturation and migration of dendritic cells 2. Granulocytic di erentiation ATP NF546 AR-C67085 ATP-γ-S AP 4A NF340 Suramin RB2 P2Y12 Platelets, glial cells, spinal cord, and glial cells 1. Platelet aggregation 2. A role in dense granule secretion 3. Maintenance of nociception sensitivity (neural-glial cell interactions) ADP 2-MeSADP ADP-β-S AZD6140 (Ticagrelor) AR-C69931MX (Cangrelor) PSB-0739 MRS MeSAMP P2Y13 Spleen, brain, lymph nodes, bone marrow, erythrocytes, liver, pancreas, and heart 1. Platelet aggregation 2. A role in dense granule secretion 3. P2Y13 receptors are present in both immune system and brain, but its function is largely unknown ADP 2-MeSADP MRS2211 AR-C69931MX (Cangrelor) 2-MeSAMP P2Y14 Placenta, adipose tissue, stomach, intestine, discrete brain regions, spleen, lung, heart, bone marrow, peripheral immune cells, and mast cells 1. Chemoa ractant receptor in bone marrow hematopoietic stem cells 2. Dendritic cell activation UDP/UDPglucose/ UDP-galactose MRS2690 MRS2802 UDP may also act as antagonist * Low a nity. Abbreviations: CNS, central nervous system; NTS, nucleus tractus solitarius. For chemicals see Appendix: List of compounds. Adapted from Burnstock (2008); Burnstock and Sawynok (2010); Fredholm et al. (1994); Jacobson et al. (2006); Magni and Ceruti (2013); North and Jarvis (2013)

36 13 Table 6. Expression of purinergic P2X and P2Y receptors involved in pain transmission. Receptor Expression Functional receptors Neurons Dorsal root ganglion Trigeminal ganglion Dorsal horn spinal cord P2X 3 P2Y 1,2,4,6,12,13,14 P2X 3,P2X 4 P2Y 1,2,4,6 P2Y 1,2,4,6 P2X 3 P2Y 1,2,12,13 P2X 3 P2Y 1,2,4 P2Y 1,2,4(6) Satellite glial cells Dorsal root ganglion Trigeminal ganglion P2X 7 P2Y 1,12,14 P2X 7 P2Y 1,2,4,6,13,14 P2X 7 No functional studies on P2Y receptors P2X 7 P2Y 1,2,4,6,13,14 (P2Y 12 under painful conditions) Astrocytes Dorsal horn spinal cord * P2X 7 P2Y 1 P2X 7 P2Y 1 Microglia Dorsal horn spinal cord P2X 4, P2X 7 P2Y 2,6,12,13,14 P2X 4, P2X 7 P2Y 6,12,13,14 Immune system cells P2X 7 P2Y 2,6,12,13,14 P2X 7 P2Y 1,2,4,6,14 * No systematic evaluation of P2 receptor expression has been performed in spinal cord astrocytes. However, cortical brain astrocytes express a large range of P2X and P2Y receptors. Adapted from Magni and Ceruti (2013). Recent studies uncovered more details of purinergic mechanisms in migraine. Thus, it became clear that ATP and its breakdown products play an important role in the cortical spreading depression (CSD), in migraine-associated vasomotor mechanisms, and fast transmission or activation of satellite glial cells (SGC) during initiation and propagation of a migraine attack (Cieslak et al., 2015). It has been shown that after CSD, trigeminal meningeal nociceptors and central neurons increase the nociceptive ring indicating a positive coupling between the aura phase of migraine and the subsequent headache phase (Zhang et al., 2010). The intense and massive neuronal depolarisation during CSD, along with the overactivation of the glutamate N-methyl-D-aspartate (NMDA) receptors open the neuronal pannexin channels (Panx1) enabling ATP to be released (Karatas et al., 2013) and act as a potent algogen (pain inducer; Burnstock et al., 2011). In vessels, ATP controls the vascular tone via the endotheliumderivered relaxing factor (EDRF), which increases the cerebral blood ow associated with pain (Burnstock and Ralevic, 2014; Cieslak et al., 2015). SGCs contribute to nociception by a ecting the conduction of pain signals in nerve bres (Takeda et al., 2009) and supporting the pro-nociceptive sensitisation state in neuronal cell bodies in sensory ganglia (Costa and Moreira Neto, 2015) Purinergic hypothesis of migraine The involvement of the vascular system in the pathophysiology of migraine is classically associated with vasoconstriction fouled by vasodilatation (see Vascular hypothesis in section 3.2.3). The purinergic hypothesis foremost postulated the role of ATP on migraine pathophysiology in relation to vascular mechanisms, suggesting that the release of ATP via P2X receptors generates a vasospasm during the headache phase (Burnstock, 1989). Later, it was proposed that ATP activates the pro-nociceptive P2X3 receptors, in peripheral trigeminal neurons, which are implicated in transferring of migraine pain signals (Fabbre i, 2013; Fabbre i et al., 2006; Giniatullin et al., 2008; Staikopoulos et al., 2007). P2X3 receptors are clearly implicated in different types of pain (Burnstock, 2000; Chizh and Illes, 2001; Wirkner et al., 2007). Importantly, the migraine mediator neuropeptide calcitonin gene-related peptide (CGRP) can potentiate

37 14 the sensitisation of P2X3 receptors in trigeminal neurons (Giniatullin et al., 2008; Hullugundi et al., 2013; Simone i et al., 2008), via delayed upregulation of these receptors (Fabbre i et al., 2006; Giniatullin et al., 2008; Simone i et al., 2008). CGRP is a classical biomarker of migraine pathology, which can be found in saliva and in blood during migraine a acks, indicating its central role for migraine pathogenesis (Cernuda-Morollon et al., 2013; Olesen et al., 2009; Russo, 2015a). Notwithstanding the pro-nociceptive e ect that ATP exerts in the trigeminovascular system, its dephosphorylation product, adenosine, exerts a modulatory anti-nociceptive action on mechanisms of migraine (Goadsby et al., 2002a; Zylka et al., 2008). In particular, the activation of inhibitory adenosine A1 receptors can limit or prevent a migraine related pain state (Goadsby et al., 2002a). Thus, variations in the concentration level of ATP or adenosine may have diverse consequences on the equilibrium between pain and analgesia (Nair et al., 2010) P2X7 receptors in pain signalling The P2X7 subtype of ATP-gated receptors has its own speci c properties and is also involved in the modulation of pathological nociception. Studies with P2X7 knock-out (KO) mice have shown the loss of hypersensitivity to mechanical and thermal noxious stimuli, suggesting a role of these receptors in in ammatory and neuropathic pain (Goloncser and Sperlagh, 2014). Neuroin ammation basically acts as a supportive protective response to restore damaged tissues or cells, such as neurons and SGCs in the CNS, but it also promotes the development of chronic pain (Shabab et al., 2017). The extracellular nucleotide ATP acts as an endogenous signalling molecule contributing to in ammatory response via P2X7 subtype receptor, improving the clearance of a ected regions (Bours et al., 2006), but also contributing to pain signalling. In addition, during neuroin ammation, there is an upregulation of the P2X7 receptors located on immune cells (Table 5). The P2X7 subtype receptor is also expressed on glial cells, neuronal presynaptic terminals and immune cells with a monocyte-macrophage origin (Burnstock, 2016a). The activation of P2X7 receptors potentiate neuroin ammation by inducing in ammatory cytokines release, such as: interleukin(il)-1β, IL-18, IL-6 and tumour necrosis factor-α (Lister et al., 2007). The inhibition of P2X7 receptor, by producing KO mice (Chessell et al., 2005) or blocking the receptor (Donnelly-Roberts et al., 2008) with speci c antagonists, has shown a detriment in IL-1β production, resulting in the reduction of neuropathic pain ATP-gated P2X3 and P2X2/3 receptors in pain signalling The homomeric P2X3 and heteromeric P2X2/3 subtypes plays a central role in nociception, particularly of neuropathic and in ammatory pain. These receptors are primarily expressed in nociceptive sensory neurons (Wirkner et al., 2007) carrying pain information through primary a erents and reaching trigeminal sensory nuclei in the brainstem (Burnstock, 2016a). Studies have proved that application of P2X2, P2X3, and P2X2/3 receptor agonist (α,β-meatp) induces sensitivity to painful stimuli (hyperalgesia; Bland-Ward and Humphrey, 1997). Our working hypothesis is based on the fact that during a migraine a ack, extracellular ATP can activate P2X3 receptors in trigeminal nociceptive neurons. This e ect can be potentiated by the presence of the migraine mediator CGRP, as most of trigeminal neurons co-express P2X3 receptors and CGRP binding sites (Fabbre i et al., 2006). CGRP may not increase agonist sensitivity but it can have an e ect in restoring P2X3 receptors from desensitisation (Fabbre i et al., 2006; Giniatullin and Nistri, 2013) Purinergic-based drugs for migraine treatment Given the supporting evidence of the pro-nociceptive role of ATP on migraine, it is logical suggesting that purinergic antagonist could provide an anti-nociceptive e ect. Although there are no current reports of in-clinic application of such agents for treating migraine. However,

38 15 the multiple preclinical trials indicated the e ciency of ATP antagonist in diverse pain models. Di erent studies have shown a total suppression in the development of pain or modulation of allodynia (pain resulting from innocuous stimulus) in P2Y12-, P2X4-, and P2X7-KO mice (Goloncser and Sperlagh, 2014; Magni and Ceruti, 2013). These studies indicated that the suppression of certain purinergic receptors has a pharmacological potential for treating pain. Currently, there is a growing interest in the production of selective and stable antagonists for P2Y12, P2X3, P2X2/3, P2X4, and P2X7 receptors that are able to cross the blood-brain barrier (Burnstock, 2016b). These speci c agents are most promising as many of currently available in-clinic pain killers have a non-selective target (Pang et al., 2012) and develop secondary side e ects. As some purinergic receptors are widely expressed in di erent regions of the body, the usage of agonist or antagonist may trigger a nocive e ect. Drug design based on purinergic receptor speci city is detrimental. For example, drugs (i.e. ticagrelor, cangrelor, clopidogrel, and prasugrel; Table 5) based on the selective P2Y12 receptors antagonists have been developed to treat platelet aggregation, nociception, and the propitious e ect at vascular level on migraine. Nevertheless, these antagonist-based drugs can also lead to severe side e ects, such as peristalsis, or can compromise the immune system (Magni and Ceruti, 2013). As mentioned above, purines are implicated in the initiation and ampli cation of pain in migraine, particularly via neuronal P2X3 and P2X2/3 receptors, suggesting new pharmaceutical approaches for pain treatment. TNP-ATP (trinitrophenyl-substituted nucleotide) has been shown as a powerful P2X3 antagonist (Liang et al., 2004), but it can rapidly break-down in vivo (Burnstock, 2007a). The synthetic P2X3 antagonist A (synthetised by Abbo Laboratories) has an e ect in neuropathic pain depletion (Jarvis et al., 2002). Further studies reported compounds of traditional medicine, such as tetramethylpyrazine, sodium ferulate, puerarin, and lappaconitine, that inhibit P2X3 and/or P2X2/3 receptors to be able to treat pain. Similarly, P2X7 receptor antagonists have been produced to treat neuropathic and in ammatory pain. The best known P2X7 antagonists are: A (Donnelly-Roberts and Jarvis, 2007), A (Honore et al., 2006), oxidised ATP, tyrosine derivatives (KN-04 and KN-62), cyclic imides, adamantane and benzamide derivatives (Gunosewoyo et al., 2007), U73122, and U73343 (Takenouchi et al., 2005). Studies with A and A show a dose dependant antinociceptive e ect when administered systemically, where A is more e ective than A in alleviation of pain (Honore et al., 2006; McGaraughty and Jarvis, 2006). The use of P2X4 receptor antagonists, such as benzofuro-1,4-diazepin-2-ones (Synthetised by Bayer Health Care) and carbamazepine, have been recently proposed as an alternative for pain treatment (Burnstock, 2016b). Even though P2X4 receptors are expressed in trigeminal ganglia neurons, they are most important for the function of microglia (see Table 6), which are widely expressed at dorsal horn level in the spinal cord. Interestingly, certain types of hyperalgesia (i.e. morphine hyperalgesia), via P2X4 receptor activation, are associated with the release of the pro-nociceptive brain-derived neurotrophic factor (BDNF). Therefore, the blocking of BDNF-TrkB (Tropomyosin receptor kinase B) signalling can disrupt neuronal chloride homeostasis and reverse hyperalgesia states, representing a promising therapeutic approach to treat nociception (Ferrini et al., 2013) that may also be extended to the trigeminovascular system and migraine.

39 HYDROXYTRYPTAMINERGIC MECHANISMS IN MIGRAINE Hydroxytryptamine 5-hydroxytryptamine (5-HT), commonly known as serotonin (from Latin serum and Greek tonic), is a biogenic monoamine, which acts as hormone and mitogen, but mainly as a neurotransmi er in the peripheral (PNS) and central nervous systems (CNS; Mohammad-Zadeh et al., 2008) Hydroxytryptaminergic receptors Based on the International Union of Basic and Clinical Pharmacology (IUPHAR), 5-HT receptors are classi ed into seven families (see Table 7), some with characterised subtypes due to the nature of their ligand, where the vast majority of G-protein coupled receptors have seven transmembrane domains (metabotropic), except for 5-HT 3, which is a ligand-gated ion channel (ionotropic; Hoyer et al., 1994). Several studies have been conducted toward understanding the role of individual 5-HT subtypes (Barnes and Sharp, 1999) during diverse pain conditions (Bardin, 2011; Sommer, 2004), including migraine (Buzzi and Moskowi, 2005; Hamel, 2014; Saxena and Ferrari, 1992; Shields and Goadsby, 2006). The 5-HT 1 receptor is the largest subfamily of 5-HT receptors located in the PNS and CNS. This subtype has a wide variety of reported functions (Pithadia and Jain, 2009). In general, all members of the 5-HT 1 receptor subtype have been associated with the adenylate cyclase/camp system when activated. In addition, 5-HT 1 receptors have been related with the modulation of diverse body functions, including feeding behaviour, temperature regulation, and cognition. These have also been implicated to cause migraine pathology (Hamel, 2014). Despite the lack of strong evidence that associates high speci city of 5-HT 1 receptors with migraine, it is necessary to consider that guidelines for migraine headache encourage the use of 5-HT 1B/D receptor agonist-based drugs as the primary analgesic treatment (see section 3.2.4). The 5-HT 2 receptors are characterised by their ability to couple to the phospholipase C. These receptors have been associated with several physiological functions and pathologies related to the nervous system, including neurological and psychiatric disorders, smooth muscle contraction, feeding behaviour, sleep, locomotion, cerebrospinal uid exchange, and migraine. These receptors have a lower a nity for 5-HT in comparison with 5-HT 1 receptors (Hamel, 2014). The 5-HT 2B subtype is one of the most studied receptors likely involved in migraine pathogenesis. This receptor subtype can be found in endothelial cells of vessels in dura mater, smooth muscle cells, and trigeminal ganglia neurons. Studies show that the nitric oxide (NO) synthase is coupled to the 5-HT 2B receptor via a PDZ-domain. This may lead to the NO-dependent vasodilatation, promoting the extravasation of plasma proteins in cranial meninges and subsequently triggering a pain response (Segelcke and Messlinger, 2017). The 5-HT 3 receptor is the only ionotropic receptor able to mediate fast excitatory responses in peripheral and central sensory neurons (Cervantes-Duran et al., 2013; Hicks et al., 2002). 5-HT 3 receptors are expressed in primary nociceptive a erent bres in the dorsal horn and the monoaminergic nerve terminals in the descending inhibitory system (Färber et al., 2009). They are also present in the dorsal root, trigeminal, cranial, and enteric ganglia neurons (Bedford et al., 1998; Morales et al., 2001). Notwithstanding the wide distribution of 5-HT 3 receptors in di erent regions of the nociceptive pathways, their functional role on migraine nociception remains unclear. Discrepant data posits that in peripheral sensory nerve terminals, 5-HT 3 receptors play a pro-nociceptive role, whereas in spinal nociceptive transmission, these receptors can contribute to pain suppression (Färber et al., 2009; Pithadia and Jain, 2009). Thus, the role of 5-HT 3 receptors in the nociceptive trigeminovascular system remains controversial.

40 17 The 5-HT 7 receptors have been reported to participate in processes such as: regulation of circadian rhythms, modulation of neuronal activity, epilepsy, and pain (Barnes and Sharp, 1999). The 5-HT 7 receptors are coupled to adenylyl cyclase 5 via Gαs proteins (Viguier et al., 2013) and their role in migraine is li le studied. Table 7. Properties of 5-hydroxytryptaminergic receptors. Receptor Main distribution Main function Agonist Antagonist 5-HT 1 5-HT 1A CNS: Raphe nuclei and Hippocampus PNS: Limbic system and cholinergic heteroreceptor in myenteric plexus 1. 5-Hydroxytryptaminergic autoreceptor 2. Neuronal inhibition and hyperpolarisation 3. Facilitate Ach and noradrenaline release 4. Cholinergic nerve terminal in myenteric plexus 5. Hyperphagia 8-OH-DPAT Buspirone PA Ipsapirone Flesinoxan 5-CT Quetiapine WAY F 8-OH-DPAT Spiperone Sibutramine 5-HT 1B CNS: Subiculum substania nigra, basal ganglia, and striato nigral PNS: Vascular smooth muscle 1. 5-Hydroxytryptaminergic autoreceptor 2. Inhibition of neurotransmi er release 3. Terminal heteroreceptor to control release of Ach and noradrenaline 4. Contraction of vascular smooth muscle 5-CT 8-OH-DPAT Sumatriptan Ergotamine PA CP-93,129 (rodents) GR55562 SB SB Methiothepin Cynopindolol 5-HT 1D CNS: Cranial blood vessel PNS: Vascular smooth muscle 1. 5-Hydroxytryptaminergic autoreceptor 2. GABAergic and Cholinergic heteroreceptor 3. Vasoconstriction of intracranial blood vessel smooth muscle Sumatriptan Zolmitriptan Nortriptan L Ergotamine PA PNU PNU Methiothepin Ergotamine BRL HT 1E CNS: Cortex striatum PNS: mrna in vascular tissue Inhibition of adenylyl cyclase 5-HT >> 5-CT BRL54443 Methiothepin NS 5-HT 1F CNS: Spinal cord hippocampus PNS: Uterus, mesentery, and vascular smooth muscle 1. Inhibition of adenylyl cyclase 2. Trigeminal (V) neuro inhibition in guinea pig and rat LY LY Methiothepin NS 5-HT 2 5-HT 2A CNS: Cerebral cortex PNS: Gastrointestinal, vascular and bronchial smooth muscle, platelets, and lung 1. Neuro excitation 2. Broncho constriction 3. Platelet aggregation 4. Smooth muscle contraction α-methyl 5-HT 5-CT Sumatriptan 8-OH-DPAT LSD DOI DOB Ketanserin Cyproheptadin Pizoti n Methylsergide Risperidone Olanzapine Clozapine MDL HT 2B (previous 5-HT 2F ) CNS: Cerebellum hypothalamus PNS: Vascular endothelium and stomach Endothelium dependent vaso-relaxation via NO production and stomach fundus contraction 5-CT Sumatriptan BW723C86 DOI RS SB HT 2C (previous 5-H 1C ) CNS: Choroid plexus, hippocampus, and hypothalamus 1. Modulation of transferin production and modulation of CSF volume 2. Phosphoinositide turnover α-methyl 5-HT 5-CT Quipazine DOI Ro Methylsergide Olanzapine Mesulergine SB RS

41 18 5-HT 3 CNS: Area postrema PNS: Abdominal visceral a erent neuron 1. Stimulate vomiting by acting on CTZ and by vagal neuro excitation 2. Stimulate nociceptive nerve ending led to pain 2-Methyl 5-HT 5-MeOT mcpp Ondansetron Tropisetron Granisetron 5-HT 4 CNS: Hippocampus PNS: Gastrointestinal tract, heart, and urinary bladder 1. Neuronal excitation 2. Activation of Ach release in gut 3. Tachycardia 4. camp in CNS neurons 5. Increase gastrointestinal motility Mosapride Cisapride Zacopride Renzapride BIMU8 ML10302 SC53116 GR SB HT 5 5-HT 5A and 5-HT 5B CNS: Olfactory bulb and hebenula Not known 5-HT Ergotamine SB HT 6 CNS: Caudate putamen and hippocampus PNS: Superior cervical ganglia Modulation of CNS Ach release 5-MeO-T 5-HT SB SB SB Methiothepin Ro HT 7 CNS: Hypothalamus PNS: Gastrointestinal and vascular smooth muscle Smooth muscle relaxation 5-CT >> 5-HT Sumatriptan 8-OH-DPAT AS-19 SB Methiothepin Ro NS Non-selective; PA Partial agonist. Abbreviations: ACh, acetylcholine; CNS, central nervous system; CSF, cerebrospinal uid; CTZ, chemoreceptor trigger zone; PNS, peripheral nervous system; NO, nitric oxide. For chemicals see Appendix: List of compounds. Adapted from Pithadia and Jain, 2009, Hoyer et al., 1994, Carlos and Antoine e Maassen, Hydroxytryptaminergic hypotheses of migraine Initially, 5-HT was suggested as a potent physiological vasoconstrictor (Janeway, 1918). In 1948, Page and colleagues isolated and characterised 5-HT (Rapport et al., 1948a,b,c) con rming its action in smooth muscle contraction (Reid and Rand, 1952). The role of 5-HT on migraine was developed by Sicuteri (1959), who had demonstrated that the level of 5-HT was increased during migraine a acks and that 5-HT products were excreted in the urine as the 5-hydroxyindoleacetic acid (5-HIAA; Anthony et al., 1967). Moreover, evidence supported the temporary inhibition of migraine after the injection of small amounts of 5-HT to migraineurs (Anthony et al., 1967; Lance et al., 1967). Thus, several hypotheses of the origin of migraine with the involvement of a 5-hydroxytryptaminergic mechanism were formulated. The Vascular hypothesis was originally proposed by Thomas Willis (1684) and tested by Graham and Wol (1940). It has been suggested that uctuations in the diameter of cerebral arteries have implications on a migraine a ack, when the calibre of vessels is increased. Therefore, it was suggested that 5-HT acts as a vasoconstrictor, via 5-HT1 receptors (Tfelt-Hansen and Olesen, 2012), suppressing migraine activity (Appenzeller, 1991; Saxena and Ferrari, 1992). The Platelet hypothesis suggested that migraine was a blood disorder resulting from platelet abnormalities linked to 5-HT. 5-HT is stored in dense bodies in platelets. During a migraine at-

42 19 tack, platelets aggregate in plasma accompanied by a signi cant 5-HT release. Also, platelets can aggregate in response to external agents, like catecholamines and 5-HT; paradoxically the more platelets aggregate, the more readily they release 5-HT. At the end of the a ack, 5-HT returns to basal level (Hanington, 1978, 1989). The Neurogenic hypothesis proposed that the malfunction in central inhibition due to impaired 5-HT function, might prompt uncontrolled neuronal ring in the hypothalamus having implications in precursor s agents for migraine or inducing a secondary vascular e ect (Appenzeller, 1975, 1991; Blau, 1984). The Neurogenic in ammation hypothesis (vasodilatation and plasma protein extravasation) have an origin in the 1900s, when Bayliss (1901, 1923), Lewis (1927) and Krogh (1929) developed a premise that neurogenic in ammatory reactions are based on responses of sensory nerve terminals. However, it only began to gain strength when Jancsó (1960) proved that irritant substances associated with migraine pain, which exert a chemical desensitisation stimulus, can induce an in ammatory response by the neurogenic route (Jancso et al., 1967). Posteriorly, peptides ensuing pro-in ammation were identi ed (Moskowi, 1984; Saxena and Ferrari, 1992; Segelcke and Messlinger, 2017), as well as, a dramatic accretion in 5-HT level in plasma. However, it has been shown that between a acks the 5-HT level gets lower between a acks, while the 5-HIAA level secreted in urine gets higher (Ferrari et al., 1989). In the 1990s, Moskowi proposed that an increase in blood ow increases protein permeability, in which case plasma extravasation in dura mater can lead to trigeminal stimulation (Moskowi, 1990). Furthermore, Moskowi argued that the anti-migraine drug sumatriptan blocks the plasma extravasation associated with neurogenic in ammation (Moskowi, 1993; Moskowi and Cutrer, 1993). As a consequence of the heterogeneity of receptors there are still contradictory interpretations of the role of 5-HT in the nociceptive system (Saxena and Ferrari, 1992), since the 5-hydroxytryptaminergic mechanism for migraine pathology has not been entirely understood (Anthony et al., 1967; Dussor, 2014). In general, it is known that low 5-HT level turnover with high receptor sensitivity are important factors that increase the risk of having a migraine a ack (Panconesi, 2008). Furthermore, the pro-nociceptive pro le entailing 5-HT is partially mediated by the activation of perivascular nociceptive bres that innervate extracranial arteries (Koo and Balaban, 2006), whereas intracranial processes in meninges can activate meningeal nociceptive nerve bres delivering the pain signals to the central trigeminal neurons (Messlinger and Ellrich, 2001) Hydroxytryptaminergic-based pharmaceutical drugs for migraine treatment The gold standard treatment for migraine is based on the use of tryptamine-based drugs known as triptans, including zolmitriptan, sumatriptan, naratriptan, and rizatriptan. These agents are all the agonists of 5-HT 1B/1D receptors and in some cases of 5-HT 1F receptors (Dussor, 2014; Messlinger and Ellrich, 2001). Despite the use of triptans in migraine treatment, their speci c molecular and cellular mechanisms of action in the nociceptive system remain unclear (Dussor, 2014). The best recognised action of triptans is vasoconstriction (Feniuk et al., 1991), ascribed to the signi cant role that vasodilatation plays in migraine pathology (Burnstock, 1981; Spierings, 2003). Additionally, the most recent studies postulate that triptans act directly on the primary a erent trigeminal neurons (Evans et al., 2012) triggering the inhibitory e ect on nociceptive tra cking in sensory bres (Hou et al., 2001). Naratriptan also modulates the trigeminovascular transmission in the thalamus with a reversible inhibitory e ect when it is applied directly to the superior sagi al sinus, possibly by activation of 5-HT 1A and 5-HT HT1B/1D receptors (Shields and Goadsby, 2006). In sensory trigeminal neurons, triptans can inhibit the pro-nociceptive sensitive transient receptor potential cation channel subfamily V member 1 (TRPV1) receptors related to noxious activation or heat sensitivity (Evans et al., 2012). 5-HT tends to enhance and prolong the capsaicin-evoked TRPV1 receptors activity (Loyd et al., 2012) having a concomitant stimulatory e ect on calcitonin gene-related peptide

43 20 (CGRP) release (Loyd et al., 2011). Furthermore, sumatriptan is able to inhibit CGRP release in dura mater and brainstem (Amrutkar et al., 2012). Contrary to triptans, there is a large variation of 5-hydroxytryptaminergic antagonists of 5-HT proposed for migraine treatment. In general, 5-HT 2 antagonists are used for migraine prophylaxis but their use is restricted due to severe secondary side e ects (Hedner and Persson, 1988; Pithadia and Jain, 2009). Many of the compounds used for migraine prophylaxis (such as pizotifen, methysergide, cyproheptadine, amitriptyline) are antagonists of the 5-HT 2B receptor subtype (neither 5-HT 2A nor 5-HT 2C receptors), primarily acting via NO-dependent dilatation (Segelcke and Messlinger, 2017). Recently, 5-HT 3 antagonists have been raised as promising candidates to block nociception, based on the pro-nociceptive role of 5-HT 3 (as the only known 5-HT ionotropic receptor) in peripheral and central neurons in pain states (Cervantes-Duran et al., 2013; Hicks et al., 2002). 3.3 GLUTAMATERGIC MECHANISMS IN MIGRAINE Glutamate as neurotransmi er and neuromodulator in the nociceptive system Glutamate (also known as L-glutamic acid, Glu) is a non-essential amino acid and the major excitatory neurotransmi er in the central nervous system (Greenamyre and Porter, 1994; Meldrum, 2000). Glutamate is synthesised in neurons from surrounding precursors (Kai-Kai and Howe, 1991). During synaptic transmission, it is released from small size synaptic vesicles located in the presynaptic terminals. This neurotransmi er binds to a speci c glutamate receptor to induce an excitatory neurotransmission and a secondary intracellular signal transduction. The glutamatergic system is a fast-signalling system that mediates excitatory synaptic transmission by activation of ionotropic (iglur) and metabotropic (mglur) glutamate receptors (see Fig. 6). The activation of iglurs leads to Ca 2+ in ux, intracellular signalling pathways activation and vasoactive agents release, whereas the activation of neuronal mglurs leads to modulation of glutamate release, having a direct impact in postsynaptic excitability, as well as activation of presynaptic or postsynaptic second messengers that can trigger diverse intracellular cascades (Chan and MaassenVanDenBrink, 2014, see section 3.3.2). Glutamate is constantly recycled between neurons and glial cells. This interaction is known as the glutamate-glutamine cycle, which is fundamental for the glutamatergic signalling. This system is constituted by two key steps: The rst one, signal input, in which glutamate cycling occurs in a neuronal-glial interface and is mediated by diverse glutamate receptors. The other one, the glutamate signal termination, is mediated by plasma glutamate transporters, whereas the signal output is supported by vesicular glutamate transporters loading synaptic vesicles by this neurotransmi er (Gasparini and Gri ths, 2013). Glutamate, as the major excitatory neurotransmi er, plays an important role in various sensory pathways, including pain signalling. In the nociceptive system, glutamate activates second order nociceptive neurons in the dorsal horn or in the trigeminal nucleus caudalis acting on iglurs and mglurs. Also, glutamate plays a key role in ascending somatosensory a erents to the ventral posterolateral nucleus, particularly, signalling transmission of the spinothalamic tract and lemniscal pathways and in the excitation of the corticothalamic a erents (Andreou and Goadsby, 2009) Glutamatergic receptors The glutamate receptors are classi ed in two main classes: the glutamate ionotropic receptors (iglur), which are directly gated by glutamate binding and the glutamate metabotropic receptors (mglur) involving intracellular signalling pathways by releasing second messengers to the cytoplasm or by releasing G protein subunits in the membrane via heterotrimeric guanosine triphosphate-binding proteins.

44 21 Glutamate Receptors Ionotropic glutamate receptors (iglur) Metabotropic glutamate receptors (mglur) NMDA Non-NMDA Group I Group II Group III GluN1 GluN2A GluN2B GluN2C GluN2D GluN3A GluN3B AMPA GluA1 GluA2 GluA3 GluA4 Kainate GluK1 GluK2 GluK3 GluK4 GluK5 mglur1 mglur5 mglur2 mglur3 mglur4 mglur6 mglur7 mglur8 Ca²+ Na+ Na+ (Ca²+) Na+ (Ca²+) G /G₁₁ PLC G /G₁₁ PLC G /G₀ PLC Figure 6. Overview of glutamate receptor family. Two main subfamilies of glutamate receptors: ionotropic (iglur) composed of three receptor subtypes (NMDA, AMPA, and kainate) and metabotropic (mglur) constituted of three receptor subtypes (group I, group II, and group III). Glutamate gates Na + /Ca 2+ cation-permeable ionotropic receptor and activates metabotropic receptors via coupled G proteins having an effect on two signal transduction mechanisms: the activation of phospholipase C (PLC) and inhibition of adenylate cyclase (AC). Adapted from Kew and Kemp, 2005 Psychopharmacology, 179, p.5. Copyright 2005 by Springer-Verlag. The iglurs are located on neuronal and non-neuronal cells regulating di erent processes in the PNS and CNS. The iglurs have a homo-oligomeric or hetero-oligomeric structure as a product of di erent subunits combinations. Each subunit presents four important regions: 1) Extracellular amino terminal domain (ATD), in which NMDA subunits interact with allosteric modulators, regulating the receptor desensitisation. 2) S 1 and S 2 ligand binding domains (membrane associated), creating a ligand speci city between di erent classes of iglurs. 3) Ligand binding domain, providing a speci city to the ligand. 4) Intracellular carboxy-terminal domain, which is indispensable for intracellular tra cking, regulation and receptor localisation interaction with di erent sca olding and signal transduction proteins (Alexander et al., 2017b; Erreger et al., 2004). The iglurs are pharmacologically classi ed in three subtypes and named based on their ligand selectivity of the rst agonist identi ed that was able to activate them: N-methyl- D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate receptors (see Table 8). AMPA and kainate receptors are further grouped as non- NMDA receptors because of similarities in their sequence and cross-reactivity. All these receptors are permeable to cations such as Na +, K + and/or Ca 2+ (Kew and Kemp, 2005; Traynelis et al., 2010). The NMDA receptors, assembled as heteromers, are constituted principally by the GluN1, GluN2A, GluN2B, GluN2C, GluN3A or GluN3B subunits. NMDA receptors comprised by GluN1 and GluN2 subunits require two agonists. Thus, the primary agonist glutamate binds to S 1 and S 2 binding domain of GluN2 subunit, whereas the co-agonist glycine binds to the S 1 and S 2 binding domain of the GluN1 subunit (Alexander et al., 2017b). The NMDA receptor channel, apart from Na + and K +, is essentially permeable to Ca 2+ ions but it is blocked by magnesium ions (Sheng et al., 2015).

45 22 The AMPA receptors mediate the main part of fast excitatory neurotransmission, but their function depends on Ca 2+ permeability via the GluN2 receptor (Andreou and Goadsby, 2009). AMPA receptors assemble as homomers or heteromers constituted by the GluA1, GluA2, GluA3 and GluA4 subunits. Transmembrane AMPA receptor regulatory proteins (TARPs) of class I are auxiliary AMPA subunits that take part in cellular tra cking and channel gating. The kainate receptors are predominantly expressed as homomers of GluK1, GluK2 and GluK3 subunits, which are also capable to express as heteromers. The homomers of GluK4 and GluK5 subunits have high a nity to kainate, but lack of functionality. However, GluK4 and GluK5 subunits can form heteromers with GluK1-3. In general, kainate receptors require extracellular Na + and Cl - ions for their activation (Alexander et al., 2017b). Similar to kainate receptors, AMPA receptors present auxiliary subunits (neto proteins), which contribute to surface tra cking and modulation of sensitisation (Sheng et al., 2015). Table 8. Properties of ionotropic glutamate receptors. Receptor Main distribution Main function Agonist Antagonist NMDA, GluN GluN1 GluN2A CNS, kidney, heart, parathyroid gland, lymphocytes, keratinocytes, spleen, adrenal gland, ovaries, stomach, lower urogenital tract, renal pelvis, thymus, and bone Forebrain (adult), kidney (glomeruli), and carotid artery 1. Synapse re nement * 2. Respiration * 3. LTP and spatial learning * 4. Somatosensory neural pa ern formation * 5. Modulation of dopaminergic and 5-hydroxytryptaminergic system * 1. LTP and spatial learning * 2. Modulation of dopaminergic and 5-hydroxytryptaminergic system * Glutamate site: D-aspartic acid ED L- aspartic acid ED (RS)-(tetrazol-5-yl)glycine NMDA Homoquinolinic acid PA Glycine site: D-serine ED (GluN2D>GluN2C>GluN2B>GluN2A) Glycine ED (GluN2D>GluN2C>GluN2B>GluN2A) (+)-HA966 PA Glutamate site: D-aspartic acid ED (GluN2D>GluN2C=GluN2B>GluN2A) L- aspartic acid ED (GluN2D=GluN2B>GluN2C=GluN2A) (RS)-(tetrazol-5-yl)glycine (GluN2D>GluN2C=GluN2B>GluN2A) NMDA (GluN2D>GluN2C>GluN2B>GluN2A) Homoquinolinic acid (GluN2B GluN2A GluN2D>GluN2C; GluN2A PA and GluN2C PA ) Glycine site: D-serine ED (GluN2D>GluN2C>GluN2B>GluN2A) Glycine ED (GluN2D>GluN2C>GluN2B>GluN2A) (+)-HA966 PA Glycine site: 5,7-dichlorokynurenic acid GV196771A L L Glutamate site: CGP37849 LY NVP-AAM077 GluN2A>GluN2B (human), weakly selective in rat UBP141 (GluN2D GluN2C>GluN2A GluN2B) Conantokin-G (GluN2B>GluN2D=GluN2C=GluN2A) d-ap5 d-ccpene (GluN2A=GluN2B>GluN2C=GluN2D) Selfotel Glycine site: 5,7-dichlorokynurenic acid GV196771A L L701324

46 23 GluN2B Forebrain (postnatal), CNS (embryonic mouse), hearth (postnatal), RAECs, and kidney (cortex) 1. Neonatal death * 2. Synapse re nement * 3. Synaptic plasticity and learning * Glutamate site: D-aspartic acid ED (GluN2D>GluN2C=GluN2B>GluN2A) L- aspartic acid ED (GluN2D=GluN2B>GluN2C=GluN2A) (RS)-(tetrazol-5-yl)glycine (GluN2D>GluN2C=GluN2B>GluN2A) NMDA (GluN2D>GluN2C>GluN2B>GluN2A) Homoquinolinic acid (GluN2B GluN2A GluN2D>GluN2C; GluN2A PA and GluN2C PA ) Glycine site: D-serine ED (GluN2D>GluN2C>GluN2B>GluN2A) Glycine ED (GluN2D>GluN2C>GluN2B>GluN2A) (+)-HA966 PA Glutamate site: CGP37849 LY NVP-AAM077 GluN2A>GluN2B (human), weakly selective in rat UBP141 (GluN2D GluN2C>GluN2A GluN2B) Conantokin-G (GluN2B>GluN2D=GluN2C=GluN2A) d-ap5 d-ccpene (GluN2A=GluN2B>GluN2C=GluN2D) Selfotel Glycine site: 5,7-dichlorokynurenic acid GV196771A L L GluN2C Cerebellar granule cells (adult), kidney, heart, pancreas, skeletal muscle, RAEC, and brain (spine stellate neurons) 1. Motor function * 2. Certain synapsis Glutamate site: D-aspartic acid ED (GluN2D>GluN2C=GluN2B>GluN2A) L- aspartic acid ED (GluN2D=GluN2B>GluN2C=GluN2A) (RS)-(tetrazol-5-yl)glycine (GluN2D>GluN2C=GluN2B>GluN2A) NMDA (GluN2D>GluN2C>GluN2B>GluN2A) Homoquinolinic acid (GluN2B GluN2A GluN2D>GluN2C; Glutamate site: CGP37849 LY UBP141 (GluN2D GluN2C>GluN2A GluN2B) Conantokin-G (GluN2B>GluN2D=GluN2C=GluN2A) d-ap5 d-ccpene (GluN2A=GluN2B>GluN2C=GluN2D) Selfotel GluN2A PA and GluN2C PA ) Glycine site: D-serine ED (GluN2D>GluN2C>GluN2B>GluN2A) Glycine ED Glycine site: 5,7-dichlorokynurenic acid GV196771A L L (GluN2D>GluN2C>GluN2B>GluN2A) GluN2D Diencephalon and brainstem (postnatal), kidney (cortex), and RAECs Emotional states * Glutamate site: D-aspartic acid ED (GluN2D>GluN2C=GluN2B>GluN2A) L- aspartic acid ED (GluN2D=GluN2B>GluN2C=GluN2A) (RS)-(tetrazol-5-yl)glycine (GluN2D>GluN2C=GluN2B>GluN2A) NMDA (GluN2D>GluN2C>GluN2B>GluN2A) Homoquinolinic acid (GluN2B GluN2A GluN2D>GluN2C; GluN2A PA and GluN2C PA Glycine site: D-serine ED (GluN2D>GluN2C>GluN2B>GluN2A) Glycine ED Glutamate site: CGP37849 LY UBP141 (GluN2D GluN2C>GluN2A GluN2B) Conantokin-G (GluN2B>GluN2D=GluN2C=GluN2A) d-ap5 d-ccpene (GluN2A=GluN2B>GluN2C=GluN2D) Selfotel Glycine site: 5,7-dichlorokynurenic acid GV196771A L L (GluN2D>GluN2C>GluN2B>GluN2A) GluN3A Cortex (postnatal), kidney, and IMCDs 1. Spine morphology * 2. Possible excitatory synapsis (postnatal) Glycine site: D-serine ED Glycine ED response potentiated by Zn 2+ Glycine site: TK80 NC (GluN3B» GluN3A) TK13 NC TK30 NC GluN3B Motor neurons in brainstem and spinal cord 1. Motor coordination * Glycine site: D-serine ED Glycine ED response potentiated by Zn 2+ Glycine site: TK80 NC (GluN3B» GluN3A) TK13 NC TK30 NC

47 24 AMPA, GluA GluA1 CNS (predominantly in forebrain ** and adult brain), dorsal root ganglion, and sensory nerves 1. NMDA receptordependent LTP * 2. Spatial working memory * 3. Nociception (S)-5- uorowillardiine AMPA ATPO GYKI53655 GYKI53784 NC NBQX Tezampane GluA2 CNS (predominantly in: forebrain **, adult cortex, and cerebellar granule cells) and skin 1. Synaptic plasticity (S)-5- uorowillardiine 2. Ca 2+ permeability AMPA modulation * 3. LTP * 4. Synaptic transmission * 5. Behaviour * ATPO GYKI53655 GYKI53784 NC NBQX Tezampane GluA3 CNS and skin Synaptic transmission and plasticity in hippocampus * (S)-5- uorowillardiine AMPA ATPO GYKI53655 GYKI53784 NC NBQX Tezampane GluA4 CNS, Bergmann glia and cerebellar granule cells), retina, thalamic reticular nucleus, and forebrain (early in development) (S)-5- uorowillardiine AMPA ATPO GYKI53655 GYKI53784 NC NBQX Tezampane Kainate, GluK GluK1 GluK2 Hippocampus (postnatal), cerebellum (development), thalamus, peripheral ganglia, sensory dorsal root ganglion neurons, and sensory a erent bres Cingulate gyrus of neocortex (prenatal) and peripheral ganglia 1. Hippocampal synapses * 2. Antinociceptive responses (under pain states) 1. Seizurogenic activity * 2. LTP in amygdala (implications for contextual and auditory fear memory) * GluK3 Hippocampus Hippocampal mossy bre synapses GluK4 GluK5 Hippocampus (CA3 region, dentate gyrus, and subiculum) CNS (predominantly in hippocampus) Synaptic function in the hippocampus (S)-4-AHCP (S)-5-iodowillardiine 8-deoxyneodysiherbaine ATPA LY SYM2081 Domoic acid Dysiherbaine Kainate acid SYM2081 Domoic acid Dysiherbaine Kainate acid SYM2081 Dysiherbaine Kainate acid (low potency) SYM2081 Domoic acid Dysiherbaine Kainate acid SYM2081 Domoic acid Dysiherbaine Kainate acid Neodysiherbaine ACET LY LY MSVIII-19 NS3763 NC UBP302 UBP310 2,4-epineodysiherbaine Not-known Not-known Not-known ED Endogenous; PA Partial agonist; NC Non-competitive; * Based on studies with knock-out mice and/or impairment of functions; ** Forebrain: including hippocampus and cerebral neocortex. The CA3 region in the hippocampus receives inputs from the mossy bres (granule cells located in the dentate gyrus). Abbreviations: CNS, central nervous system; CSF, cerebrospinal uid; IMCD, inner medullary collecting duct cells; LTP, Long-term potentiation; PNS, peripheral nervous system; RAEC, rat aortic endothelial cells. For chemicals see Appendix: List of compounds. Based on (Alexander et al., 2017b; Bahn et al., 1994; Bozic and Valdivielso, 2017; Collingridge et al., 2009; Contractor and Swanson, 2008; Hashimoto, 2017; Papouin and Oliet, 2017; Petralia and Wenthold, 2008).

48 25 The metabotropic glutamate receptors (mglur) are located predominantly in the synaptic cleft, in both neurons and glial cells. Metabotropic glutamate receptors are members of the GPCR class C. The mglurs are constituted by three main groups: group I (mglur1,5), group II (mglur2,3) and group III (mglur4,6,7,8; see Table 9). Group I mglurs couple to Gq and Gq-like family of G-proteins to activate phospholipase C and release intracellular Ca 2+, while group II and group III mglurs couple to Gi and Go family of G-proteins to inhibit adenylyl cyclase and consequent cyclic adenosine monophosphate (camp) formation (Andreou and Goadsby, 2009; Hampson et al., 2008). Table 9. Properties of metabotropic glutamate receptors. Receptor Main distribution Main function Agonist Antagonist Group I mglur1 mglur5 Group II Brain, retina, nociceptive primary a erents, pancreas, heart, and thymus - postsynaptic Brain (functional role including astrocytes, presynaptic membranes, hippocampal interneurons, and glial cells), retina, mammalian optic nerve and spinal cord, nociceptive primary a erents, pancreas, heart, and thymus 1. Synaptic plasticity (induction and expression of LTP and reference memory) * 2. LTP and LTD 3. Enhance excitability 4. Prepulse inhibition 5. Encode plasticity during prolonged pain state 6. Motor control (Motor and spacial learning) * 7. Hormone release facilitation (pancreas) 8. Arterial pressure maintenance 9. Neuroprotection (cell cytotoxicity) 1. Synaptic plasticity (Maintenance and protein synthesis-dependent phase of LTP and working memory) * 2. Learning and memory 3. Motor regulation 4. Prepulse inhibition 5. Di erent signalling pathways 6. LTP and LTD 7. Processing acute nociception 8. Hormone release facilitation (pancreas) 9. Arterial pressure maintenance 10. Neuroprotection (cell cytotoxicity) 11. Elevation of intracellular calcium (S)-3,5-DHPG (S)-(+)-CBPG PA CHPG (S)-3,5-DHPG LY AIDA 3-MATIDA (S)-TBPG (S)-(+)-CBPG MCPG CPCCOEt ACDPP MCPG mglur2 CNS, pancreas, heart, and thymus - presynaptic 1. Decreasing intracellular camp and activating K + channels 2. Sense glutamate release in synaptic cleft; inhibition of presynaptic glutamate 3. Hormone release facilitation (pancreas) 4. LTD LY LY LY LY PCCG-4

49 26 mglur3 CNS, astrocytes, pancreas, heart, and thymus 1. Decreasing intracellular camp and activate K + channels 2. GABA release modulation (negatively) 3. Hormone release facilitation (pancreas) 4. Inhibition of cystine/glutamate antiporter LY LY LY LY Group III mglur4 Olfactory bulb (Granulle cells), cerebellum, and hippocampus - presynaptic 1. Inhibition of presynaptic glutamate 2. Seizure decrease and vulnerability (epilepsy) 3. Neuroprotection 4. Anxiety L-AP4 L-Serine-Ophosphate LSP mglur6 Retina Hyperpolarisation of ON-bipolar cells 1-benzyl- APDC homo-ampa mglur7 mglur8 Astrocytes, cortex, cerebellum, subcortical regions, brainstem, olfactory bulb (excl. granule cells), trigeminal and dorsal root ganglion, dorsal horn, relay nuclei of sensory pathways (high expression), limbic system, and retina astrocytes, olfactory bulb, piriform cortex, pontine grey, reticular nucleus (thalamus), forebrain **, and cerebellum 1. Neuroprotection (reduce excitotoxicity induced by glutamate); increase glutamate uptake 2. Stress 1. Seizure modulation (epilepsy) 2. Neuroprotection (reduce excitotoxicity induced by glutamate); increase glutamate uptake 3. Sensitisation (pain) L-AP4 L-Serine-Ophosphate LSP AMN082 L-Serine-Ophosphate (S)-3,4-DCPG L-AP4 MAP4 MAP4 THPG Not-known MPPG PA Partial agonist; * Based on studies with knock-out mice and/or impairment of functions; ** Forebrain: including hippocampus and cerebral neocortex. Abbreviations: camp, cyclic adenosine monophosphate; CNS, central nervous system; GABA, gamma-aminobutyric acid; NMDA, N-Methyl-D-aspartic acid; LTD, long-term depression; LTP, Long-term potentiation. For chemicals see Appendix: List of compounds. Based on (Alexander et al., 2017a; Johnson and Schoepp, 2008; Kvist et al., 2013; Li et al., 2015; Niswender and Conn, 2010; Rudy et al., 2015; Saugstad and Ingram, 2008). The mglurs are distributed throughout the CNS situated in synaptic and extra synaptic regions in neurons and glial cells (Niswender and Conn, 2010). mglurs have di erent physiological roles, however they present subgroups with similar characteristics. In general, group I mglur has an excitatory role in the postsynaptic region by acting on cell depolarisation, while group II mglur and group III mglur have an inhibitory role in the presynaptic region (Shigemoto et al., 1997). Group I mglurs stimulate Ca 2+ release from intracellular stores (vesicles and intracellular organelles; Verkhratsky and Petersen, 1998), which regulates di erent signalling pathways. The ligand causes phospholipase C (PLC) activation by coupling Gq/11 that leads to inositol 1,4,5-triphosphate (IP 3 ) and diacyl glycerol formation, via Homer proteins, allowing IP 3 to open Ca 2+ channels. The mglur1 promote a fast and transient Ca 2+ release, while mglur5 induces Ca 2+ oscillations (i.e. in astrocytes, Ca 2+ oscillations induce glutamate release). Additionally, these signalling molecules can activate other signalling pathways, such as protein kinase A (PKA) and protein kinase C (PKC), which act on p44/42 mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway and subsequently activate the camp response element-binding protein (CREB) having a secondary role in gene expression and modulation of the neuropeptide CGRP (Uematsu et al., 2015). Furthermore, the activation of mglu5 receptor can potentiate purinergic A2A receptor via ERK pathway and NMDA-mediated CREB via PKC (Saugstad and Ingram, 2008). Group II and III mglurs diminish Ca 2+ in ux and subsequent glutamate release by inhibit-

50 27 ing presynaptic Ca 2+ channels via Gβγ subunits, to prevent the formation of camp, from ATP (Hampson et al., 2008). The decrease in camp stimulate G protein inwardly rectifying potassium channels (GIRKs), activating (opening) potassium channels (Johnson and Schoepp, 2008). Group II mglurs can also be situated in the postsynaptic region to induce hyperpolarisation (Johnson and Schoepp, 2008), while group III mglurs are located predominantly in the presynaptic region, in (mglu7) or near (mglu4 and 8) the active zone of the synapse. Group III mglurs are negatively coupled to adenylyl cyclase (mglur4, 7 and 8) or positively coupled to cyclic guanosine monophosphate phosphodiesterase (mglur6). The activation of group III mglurs can lead to a depression of glutamatergic and GABAergic synaptic transmission. In addition, these receptors can modulate the release of di erent transmi ers when they act as autoreceptors (i.e. glutamate) or heteroreceptors (i.e. GABA, substance P, 5-HT, dopamine, and acetylcholine; Neugebauer, 2008). However, the glutamate-binding pocket is very highly selective for glutamate over other amino acids Glutamatergic mechanisms of migraine Glutamate is localised in regions related to migraine, such as: trigeminal ganglion, the trigeminocervical complex, and thalamus (Gasparini and Gri ths, 2013). Evidence shows that local application of glutamate can produce additional nociceptive responses in the trigeminovascular system. For example, Glutamate can excite trigeminal nucleus caudalis (TNC) neurons, increasing extracellular TNC glutamate level until generation of a noxious stimulus in the trigeminal nerve (Ramadan, 2003). The role of glutamate in migraine could manifest di erently in migraine with aura than migraine without aura. Thus, experimental approaches suggest that glutamate is implicated in the initiation of migraine with aura by triggering the cortical spreading depression (CSD) underlying aura (D Andrea et al., 2001; Fi ova and Van Harreveld, 1974; Gasparini et al., 2015, 2016; Ramadan, 2003). This is best shown in the familial hemiplegic migraine (FHM; Capuani et al., 2016; Pietrobon, 2007; Swarts et al., 2013). During CSD, glutamate leads to loss of membrane potential and alterations in ionic balance (K +, Ca 2+ and Na + ). In addition, a magnesium depletion of glutamate itself can activate NMDA channels, which can contribute to CSD. The use of NMDA antagonists has been shown to reduce CSD (Gasparini et al., 2016; Ramadan, 2003; Shatillo et al., 2015). In migraine without aura, suggestions point to an interictal de ciency in the glutamatergic system and its signalling pathways. The evidence based on genetic, biochemical and clinical approaches suggests that a hypofunction within these glutamatergic signalling pathways prompt migraine (Gasparini et al., 2016). The glutamate hypothesis started to be developed when clinical migraine manifestations were concomitant to glutamate plasma level elevation in patients during a migraine a ack (Ramadan, 2003). In summary, any disruption in the distribution, expression, and modulation of glutamate receptors and its transporters may have consequences in the concentration of glutamate that can contribute to migraine pathophysiology Glutamatergic-based pharmaceutical drugs for migraine treatment Many e orts have been a ributed to pharmacochemical treatment of pain based on modulation of glutamate and its receptors, however a widespread inhibition of glutamate receptors may cause secondary e ects restricting their potential in clinical applications (Chen et al., 2008). The multifarious role and distribution of glutamate receptors provides a certain speci- city for development of potential new drugs for treating diverse neurological conditions. The major example is the use of NMDA receptor antagonist drugs, such as ketamine (Diener et al., 2015; Kalra and Ellio, 2007), dizocilpine (MK-801; Monaghan, 2007), dextromethorphan, and quinidine (Sokolov et al., 2015; Taylor et al., 2016) for treatment of migraine. These antagonists contribute to inhibiting pain transmission by blocking c-fos expression in the

51 28 TNC. In addition, MK 801 is able to induce a dural vasodilatation and has been shown to prevent CSD (Chan and MaassenVanDenBrink, 2014). Recently, memantine has been developed as a moderate-a nity uncompetitive NMDA receptor antagonist for preventive treatment of migraine, particularly during CSD phase, with low side e ects. Memantine inhibits the prolonged-glutamate induced current and Ca 2+ in ux without fully inhibiting NMDA receptor activation (Bigal et al., 2008; Vincent Chen et al., 2008). When Ca 2+ accumulates (i.e. by overactivation of NMDA receptor), it can cause the production of toxic free radicals and initiate enzymatic processes that result in cell death (Vincent Chen et al., 2008). During last years, new pharmacological strategies have expanded the use of glutamate receptor antagonists, besides NMDA, kainate, and AMPA receptors (Chan and MaassenVan- DenBrink, 2014; Diener et al., 2015; Gasparini et al., 2015), some of which are still under study (phase III). Studies have shown that the AMPA/kainate receptor antagonist tezampanel (LY or NGX424) is able to a enuate c-fos expression in the trigeminocervical complex and block plasma protein extravasation in the trigeminal ganglion (Chan and MaassenVan- DenBrink, 2014). Toripamine is an anticonvulsant able to block Na + channels, Ca 2+ channels (L-type) and kainate receptors (Chan and MaassenVanDenBrink, 2014), but it has also been shown to be an e ective preventive drug for migraine (Bussone et al., 2005). In conclusion, the role of glutamate in migraine pathology, especially in the peripheral part of the trigeminovascular system remains not fully understood and requires further detailed exploration with modern advanced techniques. 3.4 CALCITONIN GENE-RELATED PEPTIDE CGRP structure and mechanism of ligand binding The calcitonin gene-related peptide (CGRP) is a 37 amino acid sensory peptide (Iyengar et al., 2017) which has two isoforms. First, αcgrp, derived from gene CALCA, that can be found in the PNS and CNS innervating the vasculature. Second, βcgrp, derived from gene CALCB, is mainly expressed in CNS around vessels and enteric nerves (Hay and Walker, 2017; Karsan and Goadsby, 2015; Russell et al., 2014). CGRP is synthesised in sensory ganglia, like trigeminal and dorsal root ganglia (Goto et al., 2017; Russell et al., 2014), and released at the level of peripheral terminals, contributing to di erent processes including vasodilatation, nociception, sensory processing, and in ammation (Brain et al., 1985; McCulloch et al., 1986; Van Rossum et al., 1997). In sensory nerve terminals, CGRP is stored in large, dense core vesicles (Durham and Russo, 1999) that are released via calcium-dependent exocytosis during neuronal depolarisation as a result of multifarious responses, such as stress, physical damage (i.e. axotomy), in ammation, exogenous compounds, and so on (Russell et al., 2014). Neither regulation of synthesis nor regulation of CGRP removal are entirely known. However, it has been proposed that released CGRP is reuptaken into the sensory neurons by an active peptide transport mechanism (Sams-Nielsen et al., 2001) and stored by these neurons (Durham, 2006; Messlinger et al., 2011). The receptor for CGRP is a complex G protein-coupled receptor (GPCR) known as calcitonin receptor-like receptor (CLR), which is a conventional seven transmembrane helix CGRP-like protein, and a single-pass membrane protein known as the receptor activity-modifying protein (RAMP), which is an accessory protein (see Fig. 7; Watkins et al., 2013). Furthermore, RAMP has di erent types of subgroups: RAMP1 allows a high a nity for CGRP; RAMP2, promotes high response to the related peptide adrenomedullin (AM1 receptor); and RAMP3 has a second AM receptor (AM2 receptor) partly selective to CGRP. A mature CGRP receptor is composed by the heterodimerisation of CLR and one type of RAMP (Choksi et al., 2002; Russell et al., 2014; Weston et al., 2016). In addition, the CLR/RAMP complex requires a CGRP-receptor component protein (RCP), which provides speci city to the CGRP receptor to be activated by the CGRP peptide and no other ligand (Egea and Dickerson, 2012).

52 29 Hoare (2005) proposed a ligand binding mechanism for GPCR known as the two-domain model, where GPCR includes CGRP receptors. The N-terminal activation domain of CGRP can interact with the transmembrane region of CLR, while the C-terminal binding domain is associated with the extracellular N-terminus of CLR. This extracellular N-terminus can directly interact with the C-terminal portion of ligand CGRP, as well as RAMP, creating an a nity trap, which elevates the local concentration of the CGRP-ligand onto the receptor complex, facilitating a higher binding interaction (Conner et al., 2007; Hoare, 2005). N ECL1 N ECL2 ECL3 Extracellular RAMP1 TM1 TM2 TM3 TM4 TM5 TM6 TM7 Plasma membrane 343 P C 173 K L ICL1 R ICL2 249 K I 312 ICL3 R 336 K 333 `8ᵗ Helix C Intracellular RCP Figure 7. CGRP receptor components and important residues for receptor signalling and internalisation. The CGRP receptor is formed by CLR (blue), RAMP1 (yellow) and RCP (orange). Functionally important residues are shown as single letter abbreviations. Several amino acids within the CLR C-terminus ( N400-C436) and I312 at the ICL3/TM5 junction are required for effective CGRP-mediated internalisation. Abbreviations; C, C-terminal; ECL, extracellular loop; ICL, intracellular loop; N, N-terminal; TM, transmembrane. Reprinted with modifications from "Regulation of signal transduction by CGRP receptors" by Walker et al., 2010, Trends Pharmacol Sci, 31, p.478. Copyright 2010 by Elsevier Intracellular self-regulatory signalling pathways of CGRP RAMP binding to CLR produces protein conformational changes in the plasma membrane that then has implications for ligand binding and coupling for di erent intracellular signalling pathways involving CGRP (Russell et al., 2014). The activation of the CGRP receptor increases CGRP gene transcription. Exposure to CGRP can increase CGRP-mRNA levels and the CGRPpromoter in trigeminal neurons (Zhang et al., 2007b). CGRP can activate camp which in turn activates the protein kinase A (PKA) that increases CGRP gene expression (Freeland et al., 2000). This process contributes to a self-regulatory mechanism for CGRP, with e cient coupling of the receptors to the signal transduction pathways. Furthermore, the neuronal growth factor (NGF) can increase CGRP synthesis in sensory neurons (Lindsay and Harmar, 1989) by activating the MAPK/ERK pathway, as well as activating cytokines such as tumour necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β; see Fig. 8; Park and Russo, 2010).

53 30 Activin NGF NO TNF-α IL-1β CGRP MEK1/2 p38 JNK PKC camp ERK1/2 PKA plasma membrane p Smad3 USF p CREB E-box CRE/RRE enhancer CGRP transcription nucleus Figure 8. Signal transduction pathways that activate CGRP gene transcription. Arrows indicate ligand binding to its receptor at the plasma membrane. Inside the cell, arrows with straight line represent established pathways, while arrows with dotted lines are pathways whose target transcription factors are not known. Phosphorylation of transcription factor is marked as p. Heterodimer of USF1 and USF2 are depicted with two ovals in different colours. While binding sites (shown with black boxes in CGRP gene promoter) for USF and CREB are known, one for Smad3 is not identified. The horizontal arrow indicates gene expression. NGF, nerve growth factor; NO, nitric oxide; TNF, tumour necrosis factor; IL, interleukin; PKA, protein kinase A; PKC, protein kinase C; JNK, c-jun N-terminal kinase; MEK, mitogen activated protein kinase kinase; ERK1/2, extracellular signal-regulated protein kinase; USF, upstream stimulatory factor; CREB, CRE-binding protein; CRE, cyclic AMP responsive element; RRE, ras-responsive element. Reprinted with modifications from "Genetic Regulation of CGRP and Its Actions" (p. 101) by Park and Russo. In: Hay, D. L. & Dickerson, I. M. (eds.) "The calcitonin gene-related peptide family: Form, Function and Future Perspectives", 2010, Springer Dordrecht Heidelberg London New York. Copyright 2010 by Springer International Publishing Localisation of CGRP receptors CGRP receptors are expressed in trigeminal ganglia (Eftekhari et al., 2010, 2015), trigeminal nerve nuclei, the spinal cord (Smith et al., 2002; Unger and Lange, 1991), and vascular terminations of the trigeminal nerve which can activate sensory trigeminal neurons (Ho et al., 2010). In meninges, CGRP receptors are present in various cell types, including primary trigeminal a erents that innervate the middle meningeal and the cerebral arteries (Iyengar et al., 2017), which have been associated with migraine pathophysiology (Zakharov et al., 2015; Zhang et al., 2010). In addition, CGRP receptors are also present in trigeminal ganglia neurons and SGCs. In neurons they can directly control pain transmission (Li et al., 2008), whereas CGRP receptors in SGCs are likely associated with the modulation of intracellular Ca 2+ and the initiation of neuroin ammation (Ceruti et al., 2011) The importance of CGRP for migraine CGRP ful ls a crucial role in the development of migraine. On one hand, it promotes the generation of acute pain, on the other hand, CGRP induces sensitisation in the nociceptive pathways that leads to exacerbation of pain on migraineurs (Nistri et al., 2015; Ramon et al.,

54 ). At present, there is no recognised biomarker for any primary headache, however under a migraine a ack several compounds and neuropeptides are released (Edvinsson and Goadsby, 1994; Goadsby et al., 1990). In particular, CGRP levels in plasma soar dramatically within 2 hours after the commencement of a migraine a ack (Ramon et al., 2017). CGRP levels are increased in the cranial venous out ow during genuine migraine a acks (Goadsby et al., 1990), but no other substantial variation occurs in the level of other neuropeptides such as neuropeptide Y (sympathetic activity), substance P (sensory activity), or vasoactive intestinal peptide (parasympathetic activity; Edvinsson, 2015; Ramon et al., 2017). In addition, the CGRP concentration in saliva and cerebrospinal uid has been shown to increase in acute migraine (Cady et al., 2009). Pharmacological application of i.e. sumatriptan or rizatriptan for stopping migraine a acks reduces the increased-cgrp levels back to normal (Edvinsson, 2015; Gallai et al., 1995). Similarly, CGRP receptor antagonists and antibodies directed against CGRP have been shown to cease migraine a acks (Ho et al., 2008; Villalon and Van- DenBrink, 2017). All in all, the augmentation of peripheral CGRP concentration during migraine states suggests a crucial participation of this neuropeptide in the pathophysiology of migraine and the undeviating responses of CGRP receptors under CGRP exposure in the nociceptive trigeminovascular system.

55

56 4 Aims of the study The main goal of the present study was to investigate the roles of purinergic, 5-hydroxytryptaminergic, and glutamatergic receptors in the rat trigeminal nociceptive system. According to this goal, the aims were speci ed as: 1 To determine the functional role of adenosine nucleotides in the nociception of trigeminal neurons and meningeal trigeminal nerve terminals. 2 To examine the e ect of 5-hydroxytryptamine and 5-hydroxytryptaminergic agonists in the trigeminal nociceptive system. 3 To characterise the contribution of 5-hydroxytryptamine receptor subunits in the nociception of trigeminal neurons and its meningeal terminals by the use of selective blockers. 4 To explore the action of glutamate receptor agonists in the peripheral trigeminal nociception and test the role of NMDA receptors in the activation and sensitisation of trigeminal neurons and their meningeal nerve terminals. 5 To determine whether the migraine mediator calcitonin gene-related peptide potentiates nociceptive responses in trigeminal ganglion neurons.

57

58 5 Materials and Methods The overview of methods used for this study are summarised in Table 10 Table 10. General methods overview. Methods Description Article Biochemical assays Biological model Enzymatic immunoassay Bioluminescent enzyme-coupled assay Enzyme-linked immunosorbent assay, ELISA Fluorescence-activated cell sorting, FACS Histochemistry Lead Nitrate precipitation Toluidine blue Immunostaining Immunocytochemistry Immunohistochemistry Brainstem Cranial mast cell Cranial meninges Trigeminal ganglion cells I III, IV I I III IV III III III I,III-IV I-IV Cell culture Primary trigeminal ganglia from Wistar rats I-IV Electrophysiology Action potential recording (Extracellular suction electrode) Patch clamp recording (Whole-cell) Functional In vivo imaging Calcium imaging I-IV Microscopy Confocal microscopy Fluorescence microscopy Light microscopy I-IV II-IV IV III-IV I-IV 5.1 ANIMALS All experiments were performed in accordance with the European Community Council Directive 86/609/EEC and approved by the Animal Care and Use Commi ee of the University of Eastern Finland, National Laboratory Animal Centre). Animals were housed in an environmentally controlled area with own outlet ventilation (Air change: 15 times per hour; Temperature: 21±2 C; Humidity: 55±15 % relative humidity; Light cycle: 12/12 h, lights on 7:00-19:00). Food and water were available ad libitum. 5.2 CELLS AND TISSUE PREPARATION Isolated hemiskull preparation The hemiskulls were prepared as previously described by Zakharov et al. (2015). Publication I, III-IV Wistar male rats (5 weeks) were decapitated after sedation by CO 2 inhalation. Skulls were dissected and split into halves by sagi al suture to expose the dura mater. Hemiskulls where kept in gasi ed (5% CO 2 and 95% O 2 ) arti cial cerebrospinal uid (ACSF) containing (in millimolar): NaCl, 3-5 KCl, glucose, CaCl 2, MgCl 2, NaH 2 PO 4, and NaHCO 3 at ph 7.40 for min at room temperature (RT) prior to experimental recording or staining. A detailed list of reagents is provided in Table 11.

59 36 Table 11. Main reagent specifications. Name Concentration Description Manufacturer Article α,β-meatp 20 µm P2X1/3 receptor agonist Sigma-Aldrich, USA I 5-Hydroxytryptamine, 5-HT µm 5-HT receptor agonist Tocris, UK II-III A µm P2X3 receptor antagonist Alomone Labs, IL I A µm P2X7 receptor antagonist Tocris, UK I Adenosine µm Nucleoside Sigma-Aldrich, USA I Adenosine diphosphate, ADP µm Nucleotide Sigma-Aldrich, USA I Adenosine monophosphate, AMP µm Nucleotide Sigma-Aldrich, USA I Adenosine triphosphate, ATP µm Nucleotide Sigma-Aldrich, USA I Aspartate/L-Aspartic acid, Asp 1 mm Glutamate receptors agonist Bovine serum albumin, BSA 1%-2% Block unspeci c binding site Sigma-Aldrich, USA Sigma-Aldrich, USA BzATP triethylammonium salt 30 µm P2X7 receptor agonist Tocris, UK I Capsaicin 1 µm TRPV1 receptor agonist Sigma-Aldrich, USA II-IV Calcitonin gene-related peptide, CGRP 1 µm Pain mediator PolyPeptide Laboratories, FR Collagenase I 760 U/mL Enzyme Sigma-Aldrich, USA I-IV Compound 48/80 10 µg/ml Mast cell degranulation Sigma-Aldrich, USA III Dulbecco s phosphate-bu ered saline, DPBS IV III-IV I-II, IV 1X ph regulated bu er Sigma-Aldrich, USA I,III-IV Foetal bovine serum, FBS 10% Inactivate trypsin; growth supplement Gibco Invitrogen, USA Fluo-3 AM (Acetoxymethyl ester) 5 µm Calcium indicator Life technologies, USA I-IV Glutamate/L-Glutamic acid, Glu 1 mm Glutamate receptors agonist GR hydrochloride 10 µm 5-HT 1B/D receptor antagonist Sigma-Aldrich, USA Abcam, UK Glycine, Gly µm NMDA receptor co-agonist Sigma-Aldrich, USA IV Ham s F12 nutrient mixture, F12 Nutmix Standard Standard cell culture media Ionomycin 10 µm Maximum Ca 2+ response indicator Levamisole 2 mm Alkaline phosphatase inhibitor Thermo sher Scienti c, USA Alomone Labs, IL Sigma-Aldrich, USA mcpbg 0.2 µm 5-HT 3 receptor agonist Sigma-Aldrich, USA III N-Methyl-D-aspartic acid, NMDA 100 µm NMDA agonist Sigma-Aldrich, USA IV Normal goat serum, NGS 1%, 10% Block unspeci c binding site Jackson Immunores. Lab. Inc., USA Potassium chloride, KCl 50 mm Neuronal depolarisation Sigma-Aldrich, USA I-IV Sumatriptan succinate 20 µm 5-HT 1B/D receptor agonist Abcam, UK III Trizma-maleate sucrose bu er, TMSB 40 mm Trizma, 0.25 M Sucrose ph regulator Sigma-Aldrich, USA I Trypsin 0.25 mg/ml Enzyme Sigma-Aldrich, USA I-IV Tropanyl 3,5-dichlorobenzoate, MDL µm 5-HT 3 receptor antagonist Tocris, UK III Tween20 0.5% Permeabilisation Sigma-Aldrich, USA III-IV YO-PRO-1 Iodide (491/509) 10 µm Fluorophore Invitrogen, USA I I-IV IV III I-IV I-IV I III

60 Trigeminal ganglia cultures Publication I-IV The trigeminal ganglia were dissected from young male Wistar rats (10-12 postnatal). Trigeminal ganglia cell cultures were prepared as described by Malin et al. (2007), with modi cations. Isolated ganglia were treated by using an enzymatic cocktail (collagenase type I and trypsin) under continuous shaking (850 rpm) for 15 min at 37 C and centrifuged (1000 rpm) for 5 min. Pellet was resuspended in F12 Nutmix + GlutaMAX medium supplemented with foetal bovine serum (FBS) and 1% penicillin-streptomycin, and kept at 37 C, 5% CO 2 and 95% O 2 for 48 h prior to the experiment. Publication I To obtain isolated satellite glial cell (SGC) cultures, after enzymatic cell dissociation, cells were passed through 30 µm membrane lters (PARTEC, Germany) and centrifuged (1300 rpm) for 5 min Brainstem slices The brainstem slices were prepared as previously described by (Kageneck et al., 2014; Kapelsohn, 2015), with modi cations. Publication I, III-IV Wistar male rats (P10-12) were decapitated. The brains were gently removed to preserve the medulla and kept in gasi ed (5% CO 2 and 95% O 2 ) ACSF for 30 min. The samples were placed in generic gelatine-agar for stabilisation. The medullary brainstems were dissected and cut transversally in slices (400 µm) on a blade vibrotome (Campden instruments, Swi erland) in ice-cold ACSF. Slices were rinsed with ACSF prior to sample processing. 5.3 ELECTROPHYSIOLOGY Whole-cell patch-clamp recording The whole-cell membrane currents from trigeminal ganglia neuron cultures were recorded on the mounting stage of the inverted microscope Olympus IX-70 by using the EPC-10 ampli er (HEKA Elektronik, Germany). Patch pipe es were pulled from thick-wall borosilicate capillaries (1.5 mm outer diameter, 4-5 MΩ resistance, BioMedical Instruments, Germany). Pipe es were lled with intracellular solution containing (in mm): 0.5 CaCl 2, 5 MgCl 2, 10 HEPES, 130 CsCl, 5 EGTA, 5 KATP, and 0.5 NaGTP. The extracellular bath solution contained (in mm): 152 NaCl, 5 KCl, 10 glucose, 2 CaCl 2, 1 MgCl 2, 10 HEPES at ph The cells were kept at -70 mv membrane voltage. Publication II Cell cultures were pre-treated in pairs for 2 h (control vs. treatment), without and with CGRP. 5-HT (20 µm), ATP (10 µm), capsaicin (1 µm), or NMDA (100 µm with co-agonist glycine, 30 µm) were applied for 2 s by using the fast perfusion system (Rapid Solution Changer RSC-200, BioLogic Science Instruments, France). Publication III Plain 5-HT (20 µm) or the speci c 5-HT 3 receptor agonist mcpbg (0.2 µm) were applied for 2 s. Publication IV NMDA (100 µm) was applied for 2 s. The data was analysed o -line with the FitMaster software (HEKA Elektronik, Germany) Nerve action potentials recording The action potentials (spikes) were recorded by extracellular suction electrode technique in branches of the nervus spinosus from meningeal trigeminal a erents, which are detached from the trigeminal nerves of adult rats (see Fig. 9), by using the DAM80i ampli er (band pass Hz, gain 10,000). The red polished glass microelectrodes (tip diameter: 150 µm) were lled with ACSF solution. The hemiskulls were perfused for min for stabilisation prior to recording. Publication I Signals were digitalised at 125 khz by using a NIPCI6221 data acquisition board (National Instruments, Austin, TX, USA). The spikes were determined as biphasic transients with a duration between 0.3 to 3 ms. Only spikes with a standard deviation (σ) of more than 4 from mean value were considered for the data analysis. The number of spikes per second determined the spike rate. Publication I, III-IV Spontaneous spikes were recorded as control for min prior to the application of the substance of interest:

61 38 α,β-meatp (20 µm, P2X1/3 agonist), 5-HT (20 µm), A (10 µm, P2X3/P2X2/3 receptor antagonist), adenosine (100 µm), ADP (100 µm), AMP (100 µm), ATP (100 µm), capsaicin (1 µm, TRPV1 receptor agonist), compound 48/80 (10 µg/ml), GR (10 µm, 5-HT 1B/D receptor antagonist), MDL (30 µm, 5-HT 3 receptor antagonist), NMDA (100 µm), or in a combination of two or three of these agents. The o -line data was analysed with MATLAB software (MathWorks, USA) and the KlustaKwik method (Kadir et al., 2014) for spike cluster allocation. Figure 9. Experimental setup and electrophysiology. Hemiskull showing the nervus spinosus a meningeal branch of the mandibular nerve that supplies the dura mater, innervating a region of the medial meningeal artery (MMA). The nerve was dissected and placed into the suction glass electrode. Reprinted from ''Hunting for origins of migraine pain: cluster analysis of spontaneous and capsaicin-induced firing in meningeal trigeminal nerve fibers'' by Zakharov et al.., 2015, Front Cell Neurosci, 9, p Copyright 2015 by the authors. 5.4 CALCIUM IMAGING Primary trigeminal cell cultures were loaded with the calcium (Ca 2+ ) indicator, Fluo-3 AM, in F12 Nutmix medium supplemented with 10% FBS for min at 37 C followed by incubation in basic salt solution (BSS) containing (in millimolar): 152 NaCl, 2.5 KCl, 10 glucose, 2 CaCl 2, 1 MgCl 2, 10 HEPES at ph 7.40 for min RT, to wash out unspeci c binding. Cell cultures were pre-treated in pairs for 2 h (control vs. treatment), without and with migraine mediator CGRP (Excl. Publication III). Publication I Adenosine or its nucleotides: ATP, ADP, and AMP (all 10 µm) were applied for 2 s by using the fast perfusion system, succeeding by KCl (50 mm, 2 s) to induce neuronal depolarisation to distinguish neurons from SGCs (Simone i et al., 2006), and ionomycin (10 µm) for 10 s as maximum Ca 2+ response indicator for responses calibration within the same cell. For the ATP-gated P2X7 receptor expression experiments, we tested whether trigeminal cells express functional low a nity P2X7 receptors by applying a high concentration (100 µm) of ATP or ADP. In control, the rst application of ATP or ADP was followed by a second application of the same nucleotide with a 10-min interval between applications. To test the expression of P2X7 receptors, we applied the P2X7 receptor antagonist A (3 µm) 5 min preceding the application of the second nucleotide. Publication II Plain 5-HT (20 µm) was applied for 20 s followed by KCl and ionomycin. Publication III Plain 5-HT (20 µm) was applied for 20 s and capsaicin (1 µm, TRPV1 receptor agonist) for 2 s. Publication IV Glutamate (1 mm), aspartate (100 µm), or NMDA (100 µm), all

62 39 combined with co-agonist glycine (10 µm) were applied for 20 s. When BSS was prepared in absence of magnesium it consisted of (in millimolar): 152 NaCl, 3.5 KCl, 10 glucose, 2 CaCl2, 10 HEPES at ph Images were acquired using an Olympus IX-70 microscope (Olympus corporation, Japan) using 10x objective. Images were recorded with a 12-bit cooled CCD camera (PCO-SensiCam, Germany). Fluorescence was visualised by operating an imaging set up consisting of a monochromatic light source with an excitation light wavelength of 488 nm (100 ms exposure time with 1-2 CCD binning) and an emission intensity wavelength 520 nm at a sampling frequency of 2 fps (frames per second) with a controlling and data acquisition software (Publication I TILLvisION v4.0 by T.I.L.L Photonics GmbH-FEI, Germany and Publication II-IV upgraded to Live Acquisition Software, FEI Life Sciences, Germany). 5.5 HISTOCHEMISTRY Nucleotides staining in hemiskulls Publication I The distribution of nucleotidase activity in cranial meninges was studied by enzyme histochemistry (lead nitrate precipitation) staining as described by Helenius et al. (2012), with modi cations. Exposed tissue from hemiskulls was pre-treated for 2 h in pairs (control vs. treatment), without and with CGRP (see Fig. 10) and incubated for 45 min in Trizmamaleate sucrose bu er (TMSB) supplemented with levamisole. Then, cranial meninges were incubated for 40 min with 2 mm Pb(NO 3 ) 2 and a nucleotide (200 µm, ATP, ADP or AMP) in TMSB-bu er. In blank specimens, the nucleotides were omi ed. Afterwards, 0.5% (NH 4 ) 2 S was added for 10 s and the hemiskulls were rinsed with TMSB. Meninges were gently removed from the hemiskull and mounted with Aquatex medium (Merck, Germany) on slides. Images were acquired using an Olympus BX60 microscope with an Olympus DP71 camera (Olympus corporation, Japan) using 4x (PlanC 4 /0.10) and 10x (UPlanFL 10 /0.30) objectives Mast cell staining Publication III The distribution of mast cells in cranial meninges was studied by applying Toluidine Blue (a nity for acidic components) staining. Meninges were detached from the hemiskull and perfused with ACSF for 10 min. Meningeal tissue was treated for 10 min in pairs (control vs. treatment) without and with the compound 48/80, and xed with 4% paraformaldehyde (PFA) in Dulbecco s phosphate-bu ered saline (DPBS) overnight. Then, xed tissue was rinsed with DPBS and stained with Toluidine Blue (10%, ph: 2.5) and dehydrated with increasing concentrations of ethanol (70, 80, 90 and 99%) and mounted on slides with Entellan (Sigma-Aldrich Co., Germany) mounting medium. Images were acquired using an Olympus AX-70 microscope with Olympus Colour View II camera (Olympus corporation, Japan) using 20x objective. 5.6 IMMUNOSTAINING Receptor protein expression and distribution was observed in trigeminal ganglia cells by immunocytochemistry and in Aδ bres in rat meninges by immunohistochemistry. Publication III Hemiskulls were xed in 4% PFA for 2 h, the dura mater was dissected and kept in 4% PFA for 3 h. Then, the meningeal tissue was washed 3 times with DPBS for 15 min each and incubated with 10% NGS, 2% BSA, and 0.5% Tween 20 in DPBS, ph Myelinated Aδ bres in meninges were veri ed by using monoclonal mouse anti-neuro lament light chain antibody (1:300; see Table 12) and monoclonal rabbit anti-5-ht-3a antibody (1:300) incubated overnight at 4 C. The tissue was washed 3 times in DPBS and incubated 1 h with 1% NGS, 2% BSA, 0.5% Tween 20 in DPBS, ph Secondary antibodies donkey anti-rabbit IgG Alexa Fluor

63 (1:1000, Invitrogen, USA) and goat anti-mouse IgG (1:200, Jackson Immunores. Lab. Inc., USA) were incubated for 3 h. The tissue was washed and incubated with AMCA-conjugated streptavidin (1:200, Jackson Immunores. Lab. Inc., USA) for 1 h, then rinsed and cover slipped with aqua mounting medium (Sigma, Germany). Images were acquired by epi uorescence microscopy using Olympus IX-70 microscope with an Olympus XM10 camera (Olympus corporation, Japan) using 40x objective. Publication IV To label NMDA receptors, trigeminal ganglia cells were xed in 4% PFA for 20 min. Cells were treated with NH 4 Cl (0.535 mg/ml, 10 min), 0.2% Triton (15 min), glycine (200 mm, 15 min), 2% BSA (30 min), and 1% BSA (10 min) with a double washing step with DPBS between solutions. NMDA receptor subunits were labelled by using monoclonal rabbit anti-beta tubulin III (1:1000) and mouse anti-glun2a (1:1000) or anti-glun2b (1:1000). Samples were incubated overnight at 4 C, then washed 3 times in DPBS and incubated with secondary antibodies goat anti-rabbit IgG Alexa Fluor 488 (1:150, Invitrogen, a11008) and goat anti-mouse IgG Alexa Fluor (1:150, Invitrogen, a21052) in 1% BSA for 1 h. Cell slices were rinsed and cover-slipped with Mowiol glue (Sigma-Aldrich, Germany). Images were acquired by confocal microscopy using a Leica SP5 MP (Leica Microsystems Inc., Germany) microscope using 63x (HCX APO CS 63 /1.4) objective. Table 12. Specifications of primary antibodies used for immunolabelling. Antibody Host species Dilution Speci city Code Manufacturer Anti-5-HT-3A Rabbit 1:300 5-Hydroxytryptamine receptor 3A ASR-031 Alomone Labs, IL Anti-β tubulin III Rabbit 1:1000 Microtubules ab7751 Abcam, USA Anti-GluN2A Mouse 1:1000 NMDA receptor 2A ab1555p Merck, USA Anti-GluN2B Mouse 1:1000 NMDA receptor 2B ab1557p Merck, USA Anti-Neuro lament, DA2 Mouse 1:300 Neuro lament, light chain MA Invitrogen, USA 5.7 FLUORESCENCE-ACTIVATED CELL SORTING (FACS) The expression of purine P2X7 receptors in trigeminal SGCs was determined by FACS. Publication I P2X7 receptors were detected using the BD FACSCalibur (Becton Dickinson Biosciences Instruments, USA) with the YO-PRO-1 Iodide dye (penetrating cells through the activated P2X7 receptor). SGC cultures were exposed (1 min) to the P2X7 receptor agonist BzATP and antagonist A839977, prior to measurement with FACS. The data was analysed using the BD Cell Quest Pro Software (Becton Dickinson, USA). 5.8 ENZYMATIC IMMUNOASSAY Determination of purine level Publication I To measure the basal concentration of ATP, ADP, AMP, and adenosine in trigeminal ganglia cells and cranial meninges, bioluminescent, and uorometric techniques were applied. Cells were cultured in a 24-well plate for 48 h, while meninges were obtained from freshly isolated hemiskulls. Cell cultures and meninges were treated in pairs for 2 h (control vs. treatment), without and with CGRP. Cell cultures and meninges were rinsed and kept in BSS for 20 min (in meninges) or Hank s bu ered salt solution (HBSS) containing (in millimolar): 5.4 KCl, 137 NaCl, 19 glucose, 1.3 CaCl 2, 0.44 KH 2 PO 4, and 42 NaHCO 3, ph 7.40 for 30 min (in trigeminal cell). Supernatant was collected and heat-inactivated for 5 min at 65 C to measure the purine basal level (see Fig. 10). ATP and ADP concentrations were measured by using a bioluminescent enzyme-couple assay with the ATP-lite assay kit (Perkin Elmer,

64 41 Netherlands) following manufacturer speci cations and instructions. AMP and adenosine concentrations were treated with enzymes that convert purines into H 2 O 2 and uric acid. Then, H 2 O 2 was detected uorometrically using the Amplex Red Reagent with an emission wavelength of 545 nm and an excitation wavelength of 590 nm. Measurements were acquired using an In nite M200 microplate reader (Tecan, Austria). Calibration curves were generated in each assay using exogenous purine standard solutions with known concentrations. To determine the nucleotide hydrolysis rate in meninges, in control and after exposure to CGRP, the tissue was incubated for 6 min with a nucleotide (500 µm, ATP, ADP or AMP). An aliquot was collected and heat-inactivated to measure the basal concentration of nucleotide. Dissection Wash -CGRP +CGRP 95% O₂ / 5% CO₂ 10 min min 120 min // o 65 C, 5 min Bioluminescent and fluorometric enzymecoupled purine-sensing assays Enzyme histochemistry in cranial meninges Figure 10. Schematic representation of enzymatic histochemistry and immunoassay procedure. Fresh hemiskulls were extracted from adult rats and kept in gasified (95% O 2 /5% CO 2 ) BSS up to 30 min prior to 2 h exposure to migraine mediator calcitonin gene-related peptide (CGRP). Samples were treated in pairs control vs. CGRP. Post-treatment, BSS was collected in an Eppendorf tube and heated at 65 C for 6 min to inactivate further enzymatic degradation. Meningeal tissue from hemiskulls was processed for enzyme histochemistry (see section for further details). Image credits: Gennady Yegutkin, with modifications, unpublished Determination of the calcitonin gene-related peptide level The neuropeptide CGRP released from cranial meninges, brainstem slices, or trigeminal ganglia cells was measured using the CGRP Enzyme Immunoassay kit (EIA-Rat Kit, SPIbio, France). Publication III Hemiskulls were freshly isolated and washed with oxygenated ACSF for 30 min RT and rinsed three times (15 min each). Hemiskulls were incubated with 350 µl ACSF for 15 min, then, a 250 µl aliquot was collected and placed in the Eppendorf tube with EIA bu er (EIA kit, SPIbio, France) containing a CGRP peptidase inhibitor. Hemiskulls were re lled with 250 µl ACSF and incubated for 15 min to collect a second sample. Finally, hemiskulls were re lled with 250 µl 5-HT (20 µm, nal concentration) in ACSF and incubated for 15 min, then, a third sample was collected. Brainstem slices were kept in a 96-well plate with oxygenated ACSF at 37 C in a humidi ed atmosphere (5% CO 2 /95% O 2 ) for 30 min. An aliquot (100 µl, basal level) was collected and placed in an Eppendorf tube. Fresh ACSF was added and a second sample was collected post incubation period (15 min). Posteriorly, 5-HT (20 µm, nal concentration) in ACSF was added and a sample collected after incubation (15 min). Publication IV Trigeminal ganglia cells were plated on 24-well plates and cultured for 48 h prior to experiments. Each well was washed

65 42 with DPBS (350 µl, once) and HBSS (350 µl; three times) at 37 C in a humidi ed atmosphere (5% CO 2 /95% O 2 ) for 10 min for stabilisation. Then, the substrate was removed and replaced by the vehicle substance (HBSS; for capsaicin or glutamate experimental set) or glycine (10 µm, for NMDA experimental set). Then, two samples (200 µl each) were collected with a 15 min interval and fresh HBSS was added after each collection. Lastly, wells were re lled with capsaicin (1 µm), glutamate (1 mm), or NMDA (30 µm) with glycine (10 µm) and incubated for 15 min prior to sample collection. After each sample collection, Eppendorf tubes were placed in liquid nitrogen for 20 s and kept at -20 C. Samples must reach RT prior to be tested with the enzymatic immunoassay kit. Aliquots were tested in duplicates using the CGRP (rat) EIA kit following manufacturer speci cations and instructions. Absorbance was measured using a microplate reader Wallac 1420 Victor2 (Perkin Elmer, USA) at a wavelength of 405 nm. Calibration curves were generated in each assay by using CGRP standard solutions with known concentrations. 5.9 STATISTICAL ANALYSIS Live cell imaging, electrophysiology, or enzymatic immune assay data was analysed using MATLAB R2015a (MathWorks, USA), Excel 2013/Excel 2016 (Microsoft, USA), and Fiji/ImageJ (h ps://imagej.net/fiji) softwares. The Origin 8/Origin 9 (MicroCal, USA), GraphPad Prism5 (GraphPad Software, USA), and Statistics toolbox in MATLAB were used for statistical analysis. The paired two-sided Wilcoxon signed-rank test or paired-sample t-test were used to compare data within the same preparation but exposed to di erent probes. Whereas, the unpaired Wilcoxon-Mann-Whitney test or unpaired (two sample) Student s t-test (also known as t-test) were used for comparison between di erent preparations. The results are presented as mean±sem (standard error of the mean) with the level of signi cance set as *p<0.05, **p < 0.01 or ***p <

66 6 Results 6.1 PURINERGIC MECHANISMS IN THE RAT TRIGEMINAL SYSTEM Peripheral trigeminal nerve terminal activation by ATP and its dephosphorylation derivatives in rat meninges Publication I Nociceptive spikes were recorded in nervus spinosus peripheral terminals of cranial meninges during the administration of ATP, ADP, AMP or adenosine. We observed that among all tested substances, only ATP (n=12 experiments) increased (p<0.05; Fig. 11 A-B) the nociceptive spike ring signi cantly, whereas ADP (n=11 experiments), AMP (n=6 experiments), and adenosine (n=11 experiments) were ine ective (p>0.05; Fig. 11 C-E). A B D -1 Frequency, s -1 Frequency, s control ATP * * * * Time, min AMP Time, min 0.05 mv C E 2 s -1 Frequency, s -1 Frequency, s ATP ADP Time, min 2 Ado Time, min Figure 11. Effect of adenine nucleotides and adenosine on trigeminal nerve terminals in meninges. A. Representative traces showing trigeminal nerve activity in meninges. The evoked spikes in control (left) and during ATP (100 µm) application (right). B. Time-course traces showing variations in the frequency of meningeal activity during ATP (n=12 hemiskulls) application. C. Analogous experiments during ADP (100 µm; n=11 hemiskulls), D. AMP (100 µm; n=6 hemiskulls), and E. Ado (100 µm; n=11 hemiskulls) applications. Mean±SEM, *p<0.05, determined by t-paired test. Abbreviations: ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate, Ado, adenosine. Bar nucleotide/nucleoside application The ATP-gated P2X3 receptor expression in trigeminal nerve terminals Publication I To evaluate the nociceptive action of ATP in the purinergic trigeminal nerve terminals, we explored the role of ATP-gated P2X3 receptors. To this end, we applied the P2X1/3 agonist, α,β-meatp, alone or with the P2X3 antagonist, A We showed that α,β-meatp induced a strong pro-nociceptive ring (n=5 experiments; p<0.05; Fig. 12 A-B) with a long-lasting ATP-like e ect. When A was applied 10 min before starting α,β-meatp application, the nociceptive ring ceased (n=5 experiments; p>0.05; Fig. 12 C).

67 44 A Control 0.02 mv a,b-meatp 2 s B -1 Frequency, s a,b-meatp * * Time, min C -1 Frequency, s a,b-meatp A Time, min Figure 12. Expression of P2X3 receptors in activation of trigeminal nerve terminals in meninges. A. Representative traces showing spikes in control (top) and during application of α,β-meatp (20 µm, bottom) of trigeminal nerve in meninges. B. Time-course traces showing variations in the frequency of meningeal activity during α,β-meatp (n=5 hemiskulls) application and C. P2X3 receptor antagonist, A (10 µm), 10 min prior to α,β-meatp application. Mean±SEM, *p<0.05, determined by t-paired test. Bar, α,β-meatp or A application Calcium responses evoked by ATP and its dephosphorylation derivatives in rat trigeminal ganglia cells Publication I Our aim was to explore adenine nucleotides and nucleoside ability of evoking Ca 2+ -transients in trigeminal ganglia cells. Our results show that adenosine triphosphate (ATP) and adenosine diphosphate (ADP) are highly active in trigeminal cells. When we calculated the ADP/ATP ratio in both neuronal and SGCs responses, we noticed that the ratio was three times bigger in SGCs (1.31, n=282) but remained almost unchanged in neurons (1.03, n=173). Interestingly, ADP (81.6±2.3%) had a higher response in SGCs than its parent compound ATP (62.1±2.9%, p<0.05). In contrast, adenosine monophosphate (AMP) and adenosine were almost ine ective as a fast agonist in both cell populations (Fig. 13). A Neurons ATP AMP ADP Ado KCl % of cells B SGC 30% DF/F s ATP AMP ADP Ado KCl C Neurons D SGC * ATP ADPAMP Ado 0 ATP ADPAMP Ado % of cells Figure 13. Ca 2+ -transients activated by adenine nucleotides in rat trigeminal cells. A. Representative ΔF/F 0 Ca 2+ -transient traces (n=10 traces, average) evoked by ATP, ADP, AMP (adenosine tri-, di-, and monophosphate respectively), and adenosine solutions (10 µm, 2s) in trigeminal ganglia neurons. Potassium chloride (KCl) solution (50 mm, 2s) was applied to differentiate neurons from satellite glial cells (SGC). B. Representative Ca 2+ -traces from SGCs. C. Neuronal responses to adenine nucleotides and adenosine. D. SGCs responding to adenine nucleotides. Mean±SEM. *p<0.05, determined by Mann-Whitney test The ATP-gated P2X7 receptor expression in satellite glial cells Publication I To test whether trigeminal cells express functional low a nity P2X7 receptors, we measured the intracellular Ca 2+ -evoked responses to a high concentration (100 µm) of ATP or ADP, before and after the administration of P2X7 receptor antagonist, A In trigeminal neurons, we observed no signi cant di erences between the two applications of ATP in both scenarios; with (65.7±1.6% after A839977, n=46, p>0.05) or without (73.4±3.7% in control, n=29; p>0.05; Fig. 14) A In the same manner, the control experiment showed no di erences between the two administrations of ADP; neither with (66.2±3.6%, n=26 cells; p>0.05) nor without (72.4±3.3%, n=18 cells; p>0.05) the P2X7 receptor antagonist. Interestingly, in SGCs, we observed a dramatic depression on the second response of ATP

68 45 after exposure to A (39.1±0.9%, n=120 cells, p<0.001) that we did not nd in the control (83.7±3.7%, n=70 cells; p>0.05). No di erences were observed in the ADP paradigm (50.6±6.8% in control, n=19 cells vs 40.6±4.7% after A839977, n=20 cells; p>0.05). A B C ATP ATP 2+ Ca transient, ratio, % Control Control neuron ATP ATP KCl ATP KCl *** SGC A839977: _ + _ + Neurons SGC 30% DF/F s ATP ATP 2+ Ca transient, ratio, % A839977: neuron A A ADP ATP KCl ATP KCl SGC _ + _ + Figure 14. Testing expression of P2X7 receptors in trigeminal cells. A. Representative ΔF/F 0 Ca 2+ -transient traces (n=5, average) evoked by ATP (100 μm, 2s) in control (top) and under effect of P2X7 receptor antagonist, A839977, (3 µm, bottom) in trigeminal neurons and B. in SGC. C. First to second response ratio to ATP (top) in control (n=29 neurons; n=70 SGCs) and after A (n=46 neurons; n=120 SGCs). The second response of ATP decreased significantly after application of P2X7 receptor antagonist in SGCs. Analogous application of ADP (Bottom) in control (n=18 neurons; n=19 SGCs) and after A application (n=26 neurons; n=26 SGC). Mean±SEM, ***p < 0.001, determined by the Mann-Whitney test. Abbreviations: ATP, adenosine triphosphate; ADP, adenosine diphosphate; SGC, satellite glial cells. Bar A application; -, control; +, A Furthermore, to corroborate P2X7 receptors expression in SGCs, we used uorescence-activated cell sorting (FACS). A low basal penetration, in control, was observed (3.4±0.4%, n=5 experiments), which signi cantly increased when these cells were exposed to P2X7 receptor agonist, BzATP, (10.2±1.9%, n=5 experiments; p<0.05). When cells were exposed to P2X7 receptor antagonist, A839977, combined with BzATP, these values were comparable to basal measurements (4.3±0.5%, n=5 experiments, p<0.05; Fig. 15). A Count control % 17.2% 6.7% BzATP YO-PRO1 fluorescence A Bz ATP B Gated, (YO-PRO1 positive), % * Control Bz ATP A Bz ATP * Figure 15. YO-PRO1 uptake via P2X7 receptors in trigeminal satellite glial cells. A. Representative plot showing YO-PRO1 uptake by satellite glial cells (SGC) in control (left), after application to P2X7 receptor agonist, BzATP, (30 µm, middle), and BzATP combined with P2X7 receptor antagonist, A (5 µm, right). B. YO-PRO1 uptake via P2X7 receptor (n=5 experiments). Mean±SEM, *p<0.05, determined by t-paired test. Bar, A application.

69 HYDROXYTRYPTAMINERGIC MECHANISMS IN THE RAT TRIGEMI- NAL SYSTEM Peripheral trigeminal nerve terminals activation by 5-HT in rat meninges Publication III To study the excitability of cranial meningeal trigeminal nerve terminals in the presence of 5-HT, we proceeded in a similar manner to the previous ATP experiments (see section 6.1.1) by recording nociceptive spikes from rat hemiskulls. We compared baseline spontaneous spiking activity with ring observed during and after 5-HT application to nerve terminals at di erent concentrations (0.2 µm, n=4; 2 µm, n=6; 20 µm, n=11; or 100 µm, n=13 hemiskulls). Our data showed that 5-HT (2-100 µm) induced a robust nociceptive ring (p<0.01 to p<0.001; Fig. 16) with a very long-lasting e ect that persisted up to 20 min after 5-HT application. A control 20 µm 5-HT B 800 * * control 20 µm 5-HT 10 a.u. 1 ms nerve activity 2 mv MUA, (5min),% * 5-HT wash (10 min) wash min 30 min control µm 2 µm 20 µm 100 µm 5-HT Figure HT-induced nociceptive firing in meningeal trigeminal nerve terminals. A. Representative traces showing a typical spike shape in trigeminal nerves from cranial meninges in control (top, left) and during 5-hydroxytryptamine (5-HT, 20 µm) application (top, right). Representative traces of spikes recorded, in trigeminal nerve, before (control), during, and after (wash) 5-HT application (bottom). B. Histogram showing the number of spikes recorded during 5-HT application at different concentrations (0.2 µm, n=4; 2 µm, n=6; 20 µm, n=11; 100 µm, n=13) followed by a 10 min washout period. Mean±SEM, **p < 0.01, ***p < determined by t-paired test. Plotted line, control. a.u., Arbitrary units The 5-HT receptor mediation of peripheral trigeminal nociceptive responses Our aim was to identify the 5-HT receptor subtype(s) capable of mediating a direct excitatory response on meningeal trigeminal nerve terminals. We determined whether the ionotropic 5- HT receptor subtype, 5-HT 3, able to mediate fast depolarising responses in neurons (Alexander et al., 2017b; Machu, 2011), was involved in meningeal fast excitatory responses. Publication III We started by immunocytochemically localising 5-HT 3 receptors and neuro laments in cranial meninges. Our immunolabelling shows a co-localisation between the 5-HT 3 receptor and the slightly myelinated Aδ- bres surrounding blood vessels in dura mater (Fig. 17 A). Nociceptive spikes were recorded in meningeal trigeminal nerve terminals during administration of 5-HT and the 5-HT 3 antagonist, MDL (Fig. 17 B). We observed that overall ring activity evoked by 5-HT (801±174%, n=13 experiments) largely decreased after exposure to MDL (280±70%, n=9 experiments; Fig. 17 C). In addition, to explore the involvement of other 5-HT receptor subtypes 5-HT 1B and 5-HT 1D, we applied 5-HT in combination

70 47 with the 5-HT 1B/D receptor antagonist, GR GR also diminished the ring activity (244±30%, n=6 experiments; p<0.01; Fig. 17 C) evoked by 5HT. When both antagonists, MDL and GR127935, were administered at the same time, we found a conspicuous depletion in the e ect of 5-HT (133±43%, n=5 experiments; p<0.01; Fig. 17 C). Subsequently, we conducted parallel experiments with the speci c 5-HT 3 agonist mcpbg. We obtained an increase in the overall ring activity (233±40%, n=8 experiments; p<0.01) which ceased (70±15%, n=5 experiments; p<0.01; Fig. 17 D) after MDL exposure. Since, the 5-HT 1B/D receptor antagonist (GR127935) was partially e ective, we also applied the 5- HT 1B/D receptor agonist sumatriptan to the nerve terminals. The administration of only sumatriptan evoked a negligible ring (123±25%, n=5 experiments; p>0.05; Fig. 17 D) in the meningeal trigeminal nerve terminals. A C blood vessel MUA (in 5 min), % 5-HT HT blood vessel ** ** ** NF blood vessel * 5-HT 5-HT 5-HT +MDL +MDL +GR +GR B D MUA (in 5 min), % MUA/10s MUA/10s mm MDL 20 mm 5-HT 20 mm 5-HT time, min ** ** 0 mcpbg mcpbg+mdl Suma mcpbg Suma * Figure 17. Receptors mediating the 5-HT-induced activity in the meningeal trigeminal nerve. A. Representative traces showing nociceptive spikes evoked by the application of 5- hydroxytryptamine (5-HT, 20 µm, n=13 experiments, top) and the application of 5-HT combined with 5-HT 3 receptor antagonist (MDL-72222, MDL, 30 µm, n=9 experiments, bottom) in trigeminal nerve terminals. B. Effect of MDL-72222, 5-HT 1B/D receptor blocker (GR127935, GR, 10 µm), or both, MDL and GR127935, in nociceptive spike activity evoked by 5-HT (n=5 to 13 experiments). The number of spikes were recorded for 5 min and plotted as percentage in comparison with spike activity prior to agents' application. C. Histogram showing the action of specific 5-HT 3 receptor agonist (mcpbg, 0.2 µm), mcpbg combined with MDL-72222, 5-HT 1B/D receptor agonist (sumatriptan, suma, 20 µm), or mcpbg combined with sumatriptan (n=5 to 8 experiments). D. Immunohistochemical staining of 5-HT 3A receptor (green, left) expressed in cranial meningeal sensory fibre near to blood vessel. Co-staining with anti-light chain neurofilament (NF, red, centre) antibodies showing co-localisation of 5-HT 3A receptor in a myelinated A-fibre (merged image,, right) Mean±SEM, *p<0.05, **p < 0.01 determined by t-paired test. MUA, multiple unit activity; Plotted line, control.

71 Calcium responses evoked by 5-HT in rat trigeminal ganglia cells Publication III The functional expression of 5-hydroxytryptamine (5-HT) receptors was corroborated by the ability of 5-HT to evoke Ca 2+ -transients in trigeminal neurons and SGCs. We observed that 5-HT increased the intracellular Ca 2+ -responses in neurons (by 21.4±3.3%, n=33/154) and SGCs (by 21.0±3.5%, n=29/138; Fig. 18). Additionally, we applied capsaicin, which is a transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor agonist, to identify the nociceptive sensory neurons (Caterina et al., 1997). We showed that among 5-HT-sensitive neurons, 70% (n=23/33 neurons) also responded to capsaicin (5-HT & capsaicin: ±2.88%, n=23/154). A 5-HT capsaicin KCl neurons B % of cells Neurons C % of cells SGCs SGCs 20% DF/F 0 50 s 0 5-HT caps 5-HT +caps 0 5-HT caps 5-HT +caps Figure 18. Ca 2+ -transients activated by 5-HT in rat trigeminal cells. A. Representative ΔF/F 0 Ca 2+ -traces from trigeminal neurons (top, middle) and satellite glial cells (SGC, bottom) showing intracellular Ca 2+ elevation evoked by 5-hydroxytryptamine (5-HT, 20 µm, 20s) and capsaicin (caps, 1 µm, 2s). KCl (50 mm, 2s) was applied to differentiate neurons from SGCs. B. Portion of neurons responding to 5-HT, capsaicin, or both agonists. C. The fraction of SGCs responding to 5-HT. SGCs did not respond to capsaicin Membrane currents activated by 5-HT in rat trigeminal neurons Publication II Some sensitive neurons co-express purinergic ATP-gated P2X and TRPV1 receptors (Simone i et al., 2006). Therefore, we wanted to evaluate whether 5-HT receptors also co-express with purinergic and TRPV1 receptors. By using whole-cell patch clamp technique, we found that 5-HT evoked membrane currents in 37% of neurons (see Fig. 19). Among these 5-HT-sensitive neurons, 9% responded to capsaicin, 36% to ATP, and 36% to both, ATP and capsaicin, showing a 70% leading purinergic pro le in 5-HT-sensitive neurons. The current-densities can be found in Publication II. 5-HT A B C D +caps 5-HT ATP capsaicin 5-HT 4 pa/pf 1 s 10 pa/pf 1 s 20 pa/pf 1 s 5-HT 37% 5-HT +caps +ATP 36% 9% 19% 36% 5-HT +ATP Figure 19. Membrane currents activated by 5-HT, ATP, and capsaicin in trigeminal neurons. Representative membrane currents activated by A. 5-hydroxytryptamine (5-HT, 20 µm), B. adenosine triphosphate (ATP, 10 µm), and C. capsaicin (caps, 10 µm) in trigeminal neurons. D. Percentage of current-activated responding neurons induced by 5-HT (left) and co-expression with ATP, capsaicin or both, ATP and capsaicin, (right) Cranial meningeal mast cells as 5-HT reservoirs Publication III We wanted to evaluate whether meningeal mast cells serve as an endogenous 5-HT-release source. To this end, we induced mast cell degranulation by exposing meninges

72 49 to the compound 48/80 and measured the excitability of meningeal trigeminal nerve terminals. In these nerve terminals we observed that the exposure to the compound 48/80 induced mast cell degranulation (see Fig. 7A in Publication III) and evoked a nociceptive spike ring (250±43%, n=27 experiments; p<0.001; Fig. 7B III), whereas the 5-HT 3 antagonist MDL inhibited this spike ring (n=6; Fig. 7C III). 6.3 GLUTAMATERGIC MECHANISMS IN THE RAT TRIGEMINAL SYSTEM Peripheral trigeminal nerve terminal activation by NMDA in rat meninges Publication IV To determine the possible role of NMDA receptors in peripheral trigeminal nerve terminals, we performed electrophysiological recordings from meningeal trigeminal a erents. Spontaneous discharges in control (41.1±9.8 spikes for 2 min prior NMDA, n=8 experiments; Fig. 20) were compared with those after NMDA application. We observed a moderate but statistically signi cant spike ring evoked by NMDA combined with co-agonist glycine (58.5±9.3 spikes per 2 min post-nmda; p<0.05), compared to control. The activity gradually decreased (54.0±9.0 spikes, 2-4 min post-nmda) until reaching the control baseline (6 min post-nmda, p>0.05). A control B 100 NMDA NMDA 5 mv 30 s Frequency, s * * Time, min Figure 20. NMDA-induced nociceptive firing in meningeal trigeminal nerve terminals. A. Representative traces of spikes recorded in the trigeminal nerve, before (control) and during application of N-methyl-D-aspartate (NMDA, 100 µm) combined with co-agonist glycine (30 µm). B. Time-course trace showing variations in the frequency of meningeal activity before and after exposure to NMDA (n=8 hemiskulls). Mean±SEM, *p<0.05 determined by t-paired test. Bar, NMDA application Calcium responses evoked by glutamatergic agonists in rat trigeminal neurons Publication IV Our aim was to explore the role of ionotropic glutamate receptors, particularly the functional expression of the calcium permeable NMDA receptors in the peripheral mechanisms of the trigeminovascular system. Since NMDA receptors require two agonists to be activated, we tested the activity of these receptors with a main agonist (glutamate, aspartate, or NMDA) in the presence of the co-agonist glycine. Broad spectrum glutamate receptors agonists, glutamate and aspartate, were able to elevate Ca 2+ -responses (43.6±4.0%, n=68/156 neurons to glutamate and 51.3±5.7%, n=40/78 neurons to aspartate) in trigeminal neurons. In contrast, the NMDA receptor agonist, NMDA, induced only scarce responses (9.7±2.7%, n=12/124 neurons; Fig. 21). Intracellular Ca 2+ -transients induced by aspartate (81.3±11.1%, n=40) were higher than transients induced by glutamate (53.9±5.3%, n=68; p<0.01) and NMDA (46.8±5.4%, n=12; p=0.052) Membrane currents activated by NMDA in rat trigeminal neurons Publication IV To further characterise NMDA induced responses mediated by NMDA receptors, we implemented whole-cell patch clamp recordings from isolated trigeminal neurons. After local application of NMDA (with co-agonist glycine), we observed that NMDA evoked membrane currents in 37% (n=110 tested cells) of neurons (Fig. 22 A-B). Interestingly, most of these responses presented a low current-amplitude (Fig. 22 C).

73 50 A neuron Glu KCl C neuron NMDA capskcl E neuron Glutamate NMDA Aspartate B SGC Glu D SGC 20% DF/F s KCl 20% DF/F s NMDA caps KCl F SGC 20% DF/F s Figure 21. Ca 2+ -transients activated by glutamate, aspartate, and NMDA in rat trigeminal cells. Representative ΔF/F 0 Ca 2+ -traces from trigeminal neurons (n=5 traces, average) showing intracellular Ca 2+ -elevation evoked by A. Glutamate (Glu, 1 mm, 2 s), B. aspartate (Asp, 100 µm, 20 s), and C. NMDA (100 µm, 20 s) in adult rat trigeminal neurons. All combined with co-factor glycine (10 µm). KCl solution (50 mm, 2s) was applied to differentiate neurons from SGCs. D. Percentage of neurons responding to glutamate (n=156), aspartate (n=78), and NMDA (n=124). E. Intracellular neuronal Ca 2+ -transients evoked by glutamate (n=68), aspartate (n=40), and NMDA (n=12). Mean±SEM, **p<0.01, ***p<0.001 determined by Mann Whitney test. caps, capsaicin. Asp G 60 % of cells KCl Neurons *** *** Asp H 60 % of cells KCl SGC * *** *** 0 Glu NMDA Asp 0 Glu NMDA Asp A NMDA B C pa 1 s NMDA 37% Number of responses Current amplitude, na Figure 22. Membrane currents activated by NMDA in trigeminal neurons. A. Representative membrane-current activated by N-methyl-D-aspartate (NMDA, 100 µm, 2 s) combined with coagonist glycine (30 µm) in trigeminal neurons. B. Percentage of current-activated responding neurons induced by NMDA. C. Distribution of peak amplitudes of NMDA-evoked currents in trigeminal neurons Glutamate-NMDA receptor subtypes localisation Publication IV Subsequently, by using speci c antibodies, we immunocytochemically tested the expression of the main NMDA receptor subunits, such as the synaptic type GluN2A

74 51 and the extrasynaptic type GluN2B in trigeminal ganglia cultures. Our immunocytochemistry data showed that the GluN2A receptor subunit was expressed in trigeminal neurons and SGCs (Fig. 23 A), whereas the GluN2B receptor subtype was expressed only in neurons (Fig. 23 B). neuronal β-tubulin III GluN2A merge A neuronal β-tubulin III GluN2B merge B 50 µm Figure 23. Expression of GluN2A and GluN2B receptor subunits in trigeminal ganglia cells A. GluN2A immunostaining. B. GluN2B immunostaining. Left column, labelling of neurons with neuronal β-tubulin III (red); Centre column, labelling NMDA receptor subunits GluN2A or GluN2B (green); Right column, merged images. Magnification X63. Scale bar, 50 μm. 6.4 CGRP SENSITISING EFFECT IN RAT MENINGES AND TRIGEMINAL CELLS The e ect of CGRP on extracellular purine homeostasis Publication I Our objective was to determine the basal levels of extracellular purine (ATP, ADP, AMP, and adenosine) and their variations in migraine-like conditions (exposure to calcitonin gene-related peptide, CGRP) in cell cultures and meninges. In both scenarios, trigeminal cell cultures and meninges, ATP (in trigeminal cells: 0.96±0.2 nm in control, n=27 vs. 1.8±0.5 nm after CGRP, n=27; p<0.05; in meninges: 7±1.6 nm in control vs. 15.2±3.1 nm after CGRP, n=22; p<0.01; Fig. 24) and ADP (in trigeminal cells: 1.9±0.6 nm in control, n=27 vs. 4.7±1.2 nm after CGRP, n=27, p<0.05; in meninges: 12.7±2.4 nm in control vs. 21.6±3.3 nm after CGRP, n=22, p<0.05) levels were increased via exposure to the migraine mediator CGRP. Compared to ATP and ADP e ects, AMP and adenosine have di erent responses. On one hand, the high level of AMP did not change when exposed to CGRP in trigeminal cells (282.8±41.0 nm in control, n=19 vs ±27.4 nm after CGRP, n=20, p>0.05), but increased in meninges (375±55 nm in control vs. 600±102 nm after CGRP, n=15, p<0.05); on the other hand, adenosine decreased when exposed to CGRP in trigeminal cells (224.5±37.3 nm in control, n=19 vs ±28.3 nm after CGRP, n=20, p<0.05), but did not change in meninges (71.4±15.4 nm in control vs. 76.6±18.1 nm after CGRP, n=20; p>0.05).

75 52 A Purine levels, nm * CGRP: _ ATP trigeminal cells * ADP _ AMP _ * Ado _ + B Purine levels, nm ** 0 CGRP: _ ATP * ADP _ + meninges * AMP _ Ado _ + Figure 24. The effect of CGRP on basal purine level. The basal level of adenine nucleotides (ATP, ADP, AMP) and adenosine were measured in control and in CGRP (1 µm, 2h). A. In trigeminal neurons and B. In cranial meninges. Mean±SEM, *p<0.05, **p<0.01 determined by Student's t test (paired, two-tailed). Abbreviations: ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate, Ado, adenosine; CGRP, calcitonin gene-related peptide; -, control; +, CGRP The e ect of CGRP on nucleotide hydrolysis in rat dura mater Publication I With the goal of assessing the ectoenzymatic activity of adenine nucleotides in the cranial meningeal tissue, in physiological and under migraine-like conditions. 6 mins after exogenously applied ATP, ADP, and AMP to hemiskull preparations, we measured the level of these nucleotides. Although a decreased level was observed for all adenine nucleotides under physiological conditions (ATP: 365.3±23.4 µm, n=10; ADP: 292.6±62.1 µm, n=4; AMP: 344.4±45 µm, n=4; Fig. 25), we did not nd any di erence in the enzymatic activity in CGRPtreated samples (ATP: 377±41.9 µm, n=6; ADP: 309.5±14.6 µm, n=6; AMP: 402.3±13.8 µm, n=6; all p>0.05). Substrate, µm added purines 0 ATP _ CGRP: + ADP _ + AMP _ + Figure 25. Effect of CGRP on the rate of nucleotide hydrolysis in rat dura mater. Control and CGRP-treated (1 µm, 2h) rat hemiskulls were incubated for 6 min with an initial concentration (500 µm; dotted line) of nucleotide adenosine triphosphate (ATP), adenosine diphosphate (ADP), or adenosine monophosphate (AMP). Aliquots of the medium were collected and tested for residual concentrations of exogenously applied nucleotides. Mean±SEM determined by Student's t test (paired, two-tailed), n=8-12 hemiskull preparations. Abbreviations: ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate, CGRP, calcitonin gene-related peptide; -, control; +, CGRP. The nucleotide degradation was determined by analysing the distribution of ecto-nucleotidase-hydrolysing enzyme activity, using lead nitrate based enzyme histochemistry. A high ATPase, ADPase, and AMPase activity was found especially in the regions surrounding blood vessels belonging to cranial meninges (Fig. 26).

76 53 ATP ADP 200µM AMP 200µM Blank 200µM - Control CGRP 1µM Figure 26. Distribution of nucleotidase activity in rat dura mater. A. Rat hemiskulls in control and CGRP-treated (1 µm, 2h) were subjected to enzyme histochemical staining with Pb(NO3 )2 (2 mm, 40 min) and to an adenosine nucleotide (all 200 µm): ATP, ADP, or AMP. Control was treated in the absence of nucleotide. Nucleotidase activity shown by lead precipitation (dark spotting) is highly distributed around blood vessels and mast cells. Abbreviations: ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; CGRP, calcitonin gene-related peptide. Scale bar, 300 μm The e ect of CGRP on adenine nucleotide responses in trigeminal cells A B Control C CGRP 30% DF/F0 10 s ATP KCl ION ATP KCl ION Ca2+ transient, % Publication I Following the initial assessment of adenine nucleotides in trigeminal ganglia cells (see section 6.1.3), we evaluated the changes of ATP and ADP in these cells after the exposure to the migraine mediator CGRP. In trigeminal ganglia neurons we observed that in the presence of CGRP a small concentration (1 µm) of ATP was able to evoke responses (106.5±8.4%, n=40 in control vs ±8.7%, n=84 after CGRP; p<0.05) whereas ADP was not (24.5±2.4%, n=40 in control vs. 30.9±3.7%, n=84 after CGRP; p>0.05; Fig. 27). On the contrary, in SGCs, CGRP evoked a negligible (0%, n=82) intracellular Ca2+ elevation in both adenine nucleotides, ATP and ADP, at a very low concentration (1 µm), despite the fact that ATP-gated receptors are expressed in SGCs (see section 6.1.4). 150 * ATP CGRP: _ + _ADP+ Figure 27. The effect of CGRP on ATP- and ADP-induced intracellular Ca2+ -transients in trigeminal neurons. A. Representative ΔF/F0 Ca2+ -traces from trigeminal neurons (n=5, average) showing Ca2+ -transients evoked by adenosine triphosphate (ATP, 1 μm, 2s) in control, and B. after CGRP treatment (1 μm, 2h; n=5 traces, average). KCl (50 mm, 2s) was applied to differentiate neurons and SGC. Ionomycin (ION, 10 μm, 10s) was used for normalisation. C. The effect of CGRP on Ca2+ -transient amplitudes activated by ATP or adenosine diphosphate (ADP, 1 μm, 2s) in control and in CGRP-treated neurons. Mean±SEM, *p<0.05, determined by the Mann-Whitney test The e ect of CGRP on 5-HT responses in trigeminal cells We previously demonstrated that 5-HT evokes intracellular Ca2+ -responses in trigeminal neurons (see section 6.2.3), therefore we proceeded to test whether sensitisation by CGRP exposure has an e ect on these Ca2+ -responses. Publication II 5-HT-sensitive neuronal responses signi cantly increased from 18% (n=28/155 in control) to 35% (n=35/100 after CGRP; p<0.05; Fig. 28 A-C) after exposure to neuropeptide CGRP. Furthermore, the amplitudes of intracel-

77 54 lular Ca 2+ -responses in CGRP-treated trigeminal neurons were also elevated (26.2±2.4%, n=28 in control vs. 39.9±4.6%, n=35 after CGRP; p<0.05; Fig. 28 C). A 5-HT control ION B 5-HT CGRP ION 30% DF/F 0 10 s C % of cells ** 0 _ 5-HT CGRP: + D 2+ Ca transient, % * 0 _ 5-HT CGRP: + Figure 28. The effect of CGRP on 5-HT-induced intracellular Ca 2+ -transients in trigeminal neurons. A. Representative ΔF/F 0 Ca 2+ -traces from trigeminal neurons (n=5, average) showing Ca 2+ -transients evoked by 5-hydroxytryptamine (5-HT, 20 µm, 20 s) in control, and B. after preincubation with CGRP (1 µm, 2h). Ionomycin (ION, 1 µm, 10s, ) was used for normalisation. C. In control and CGRP-treated neurons responding to 5-HT (20 µm). D. Amplitudes of Ca 2+ -transients' responses in neurons induced by 5-HT in control and after CGRP. Mean±SEM, *p<0.05, **p<0.01, determined by the Mann-Whitney test The e ect of CGRP on NMDA responses in trigeminal cells Publication IV We showed that NMDA was able to evoke Ca 2+ -responses in trigeminal cells via NMDA receptors (see section 6.3.2). Then, we wanted to evaluate the e ect of the migraine mediator CGRP on neuronal NMDA responses. A statistically signi cant di erence (p<0.05) was found in the number of neurons responding to NMDA after 2 h exposure to CGRP (n=28/318) compared to control (n=11/247; Fig. 29 A). The amplitudes of the intracellular Ca 2+ -transients in CGRP-treated neurons were unchanged (69.4±14.6% in control, n=11/247 vs 47.6±4.7% after CGRP, n=28/318; p>0.05; Fig. 29 B). A % of cells CGRP: * NMDA _ + B 2+ Ca transient, % CGRP: NMDA _ + Figure 29. The effect of CGRP on NMDAinduced intracellular Ca 2+ -transients in young rat trigeminal neurons. A. Histogram showing portion of neurons responding to NMDA (100 µm) in control and CGRP-treated (1 µm, 2h). B. Histogram showing intracellular Ca 2+ - transients. Mean±SEM, *p<0.05 determined by Mann-Whitney test. Abbreviations: NMDA, N-methyl-D-aspartate; CGRP, calcitonin generelated peptide; -, control; +, CGRP CGRP release in rat trigeminal cells, the dura mater and the brainstem To determine the ability of di erent agonists of the trigeminovascular system to induce endogenous CGRP release, we measured the CGRP level in trigeminal neuron cultures, cranial meningeal preparations, or brainstem slices using a standard CGRP enzyme immunoassay kit. Publication III We measured the 5-HT-induced endogenous CGRP release in the PNS (cranial meninges) and CNS (medullary brainstem). In meninges, the administration of 5-HT increased the CGRP release (20±1.7 pg/ml in control vs. 34±2.9 pg/ml after 5-HT, n=5 experiments, p<0.01; Fig. 30 A), but reduced the CGRP release in the brainstem (4.32±0.70 pg/ml in control vs. 2.43±0.69 pg/ml after 5-HT, n=5 experiments, p<0.05; Fig. 30 B). Additionally, to corroborate that peripheral responses in meninges were mainly a ributed to 5-HT 3 receptors, we applied MDL (5-HT 3 antagonist) to repress the action of 5-HT. Indeed, 5-HT-