UNIVERSITÀ DEGLI STUDI DI MILANO Facoltà di Farmacia Dipartimento di Scienze Farmacologiche Corso di Dottorato di Ricerca in Scienze Farmacotossicologiche, Farmacognostiche e Biotecnologie Farmacologiche (XXIII CICLO) Graduate School in Pharmacological Sciences / Scuola di Dottorato in Scienze farmacologiche TESI DI DOTTORATO DI RICERCA PURINERGIC TRANSMISSION IN MIGRAINE: ROLE OF P2Y RECEPTORS IN THE SPINAL-TRIGEMINAL SYSTEM IN VIVO AND IN VITRO BIO/14 Tesi di dottorato di: GIOVANNI VILLA MATRICOLA: R07517 TUTOR: Chiar.ma Prof.ssa Maria Pia ABBRACCHIO CORRELATORE: Dr.ssa Stefania CERUTI COORDINATORE: Chiar.mo Prof. Guido FRANCESCHINI ANNO ACCADEMICO 2009/2010 Index INDEX 1. INTRODUCTION _______________________________ 1 1.1 PAIN AND NOCICEPTION 2 1.1.1 Molecular basis of nociception 3 1.1.2 The trigeminal nerve and the spinal-trigeminal system 4 1.1.3 Role of non-neuronal cells in pain transmission 9 1.2 MIGRAINE 12 1.2.1 Description of the migraine attack 14 1.2.2 How and where does the migraine attack originate? 15 1.2.3 Familial hemiplegic migraine 21 1.2.4 Current and future pharmacological treatment of migraine 24 1.3 THE PURINERGIC SYSTEM 29 1.3.1 Purinergic signalling 30 1.3.2 P2X receptors 32 1.3.3 P2Y receptors 33 1.3.4 Pathophysiological roles of extracellular nucleotides in the nervous system 36 1.4 PURINES AND PAIN 41 1.4.1 Role of P2X receptors in pain transmission 42 1.4.2 Role of P2Y receptors in pain transmission: sensory ganglia 46 1.4.3 Role of P2Y receptors in pain transmission: CNS 48 2. AIM OF THE STUDY ____________________________ 51 3. METHODS ____________________________________ 55 3.1 CELL CULTURES 56 3.2 PHARMACOLOGICAL TREATMENTS 58 3.3 IMMUNOCYTOCHEMISTRY 58 3.4 IMMUNOCYTOCHEMISTRY 59 Index 3.4.1 Tissue processing 59 3.4.2 Immunostaining of tissues 59 3.4.3 Quantification of results and data analysis 60 3.5 INTRACELLULAR CALCIUM MEASUREMENTS 61 3.6 TOTAL RNA ISOLATION AND RT-PCR ANALYSIS 62 3.7 WESTERN-BLOTTING ANALYSIS 65 3.8 ANALYSIS OF CGRP RELEASE BY ENZYME IMMUNO-ASSAY (EIA) 65 3.9 IN VIVO EXPERIMENTS 66 3.9.1 Animals 66 3.9.2 Orofacial formalin test 66 3.9.3 Synthesis of dsRNAs and their injection into the trigeminal ganglion 67 3.9.4 Induction of TMJ inflammation 67 3.9.5 Behavioral tests 68 3.9.6 Measurement of Evans’ blue dye extravasation 68 3.10 STATISTICAL ANALYSIS 69 4. RESULTS - Section I: in vitro studies ________________ 70 4.1 SET UP OF PRIMARY MIXED TRIGEMINAL CULTURES AS AN IN VITRO MODEL FOR STUDYING CELL TO CELL COMMUNICATION IN THE TRIGEMINAL GANGLIA 71 4.2 BOTH TRIGEMINAL NEURONS AND SATELLITE GLIAL CELLS BEAR FUNCTIONAL P2 RECEPTORS 74 4.3.. CHRONIC APPLICATION OF THE PRO-INFLAMMATORY MEDIATOR BRADYKININ DIFFERENTIALLY AFFECTS NEURONAL P2X3 AND GLIAL P2Y RECEPTOR FUNCTIONALITY 79 4.4 FOLLOWING BK APPLICATION, CGRP RELEASED FROM TG NEURONS IS RESPONSIBLE FOR P2Y RECEPTORS UPREGULATION IN SGCs 82 4.5 CGRP, BUT NOT BK, RETAINS ITS ABILITY TO INDUCE P2Y RECEPTOR POTENTIATION IN PURIFIED SGCs CULTURES 84 4.6.. THE ERK1/2 MAP KINASE PATHWAY HAS A PRIMARY ROLE IN CGRP- INDUCED POTENTIATION OF GLIAL P2Y RECEPTORS 86 4.7 CGRP RELEASE IS SIGNIFICANTLY ENHANCED IN TG CULTURES FROM CaV2.1 α1 R192Q MUTANT KNOCK-IN MICE 87 4.8.. APPLICATION OF BK INCREASES THE NUMBER OF ADP- AND UTP- RESPONDING SGCs IN TG CULTURES FROM CaV2.1 R192Q KI MICE 88 Index 4. RESULTS - Section I: in vivo studies ________________ 91 4.9 SET UP OF IN VIVO MODELS OF TRIGEMINAL PAIN FOR STUDYING ROLE OF THE PURINERGIC SYSTEM IN PAIN TRANSMISSION 92 4.10 SGCs AND MACROPHAGES ARE SELECTIVELY ACTIVATED IN TG FOLLOWING TMJ INFLAMMATION 95 4.11.. MICROGLIAL CELLS, BUT NOT ASTROCYTES, ARE ACTIVATED IN THE SPINAL TRIGEMINAL NUCLEUS FOLLOWING TMJ INFLAMMATION 98 4.12 THE PURINERGIC P2Y12 RECEPTOR IS SELECTIVELY EXPRESSED BY MICROGLIAL CELLS IN THE CNS, BUT IT IS NOT UPREGULATED BY TMJ INFLAMMATION 101 5. DISCUSSION __________________________________ 103 5.1 PRIMARY MIXED TG CULTURES AS AN IN VITRO MODEL FOR EVALUATING P2 RECEPTOR EXPRESSION AND FUNCTIONALITY 104 5.2 EXPOSURE TO ALGOGENS EXERT A COMPLEX MODULATION OF P2 RECEPTOR FUNCTIONALITY IN NEURONS AND GLIAL CELLS 107 5.3.. THE GAIN-OF-FUNCTION MUTATION IN CaV2.1 CALCIUM CHANNELS AFFECTS P2Y RECEPTORS FUNCTIONALITY IN SGCs 108 5.4.. SET UP OF IN VIVO MODELS OF TRIGEMINAL PAIN AS TOOLS FOR EVALUATING THE ROLE OF GLIAL P2Y RECEPTORS IN PAIN TRANSMISSION 110 6. REFERENCES _________________________________ 115 7. ABBREVIATIONS ______________________________ 132 1. INTRODUCTION Introduction 1.1 PAIN AND NOCICEPTION Pain can be defined as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage” (Merskey & Bogduk, 1994). It is a physical sensation arising from parts of the body, but it is also always unpleasant and therefore become an emotional experience. Experiences which resemble pain but are not unpleasant, e.g., pricking, should not be called pain (Merskey & Bogduk, 1994). Pain is a submodality of somatic sensation like touch and pressure, and serves as an important protective function by warning of injury that should be avoided or treated. However, unlike other somatic submodalities, and unlike vision, hearing, and smell, pain has an urgent and primitive quality, which is responsible for the affective and emotional aspects of pain perception (Basbaum & Jessell, 2000). The concept of pain is further complicated by the fact that its perception is always subjective. In fact, under similar conditions the same stimulus can produce different responses in different individuals (Merskey & Bogduk, 1994). Moreover, many people report pain in the absence of tissue damage or any likely pathophysiological cause; usually this happens for psychological reasons. The highly individual and subjective nature of pain makes difficult to treat it clinically. There are no “painful stimuli” that invariably elicit the perception of pain in all individuals. For example, many wounded soldiers do not feel pain until they are safely removed from battle. Similarly, athletes often do not detect their injuries until their game is over (Basbaum & Jessell, 2000). Pain can be subdivided in physiological pain and pathological or clinical pain. Physiological pain (also called acute pain, sometimes referred to as ‘‘good’’ pain) is adaptive, transient, and has a protective role that warns of potential tissue damage in response to a noxious stimulus. Pathological pain, or clinical pain (also called chronic, ‘‘bad’’ pain) is usually maladaptive, persistent, and serves no meaningful defensive, or other helpful purpose (Cao & Zhang, 2008). This kind of pain is mainly subdivided into neuropathic pain, i.e. pain associated with damage or dysfunction of the peripheral nervous system (PNS) and central nervous system (CNS), and inflammatory pain, i.e. pain related to peripheral tissue damage/inflammation (e.g. arthritic pain). In addition, other types of pathological pain, such as cancer pain, and pain elicited by continuous infusion of morphine, share some features with inflammatory and neuropathic pain but also have their distinct characteristics (Brennan et al., 1996; Mantyh et al., 2002). 2 Introduction Pathological pain is typically characterized by hyperalgesia (increased responsiveness to noxious stimuli) and allodynia (painful responses to normally innocuous stimuli), as well as by spontaneous pain. Pain hypersensitivity is not only produced in the injured tissue or territory (innervated by the injured nerve), but also spread to the adjacent non- injured regions or the extraterritory (extraterritorial pain) and to the contralateral body (mirror-image pain). This exaggerated pain is thought to result from peripheral sensitization (increase in sensitivity of nociceptive primary afferent neurons) and central sensitization (hyperexcitability of nociceptive neurons in the CNS (Cao & Zhang, 2008). In the following paragraphs the basic principles of nociception, as well as the nociceptive neuronal pathways associated to the trigeminal perception of pain, will be discussed. Finally, the emerging role(s) of CNS and PNS glial cells in pain genesis and maintenance will be analyzed. 1.1.1 Molecular basis of nociception Nociception is the process by which intense thermal, mechanical, or chemical stimuli are detected by a subpopulation of peripheral nerve fibers, called nociceptors (Basbaum et al., 2009). The cell bodies of nociceptors are located in the dorsal root ganglia (DRG) for the body, and the trigeminal ganglia (TG) for the head district, and have both a peripheral and central axonal branch that innervates their target organ and the spinal cord/brainstem, respectively (Lazarov, 2002). There are two major classes of nociceptors (Meyer at al., 2008). The first includes medium diameter myelinated (Aδ) afferents that mediate acute, well-localized “first” or fast pain (conducting signals at about 5-30 m/s). These myelinated afferents differ considerably from the larger diameter and rapidly conducting Aβ fibers that respond to innocuous mechanical stimulation (i.e., light touch). The second class of nociceptor includes small diameter unmyelinated “C” fibers that convey poorly localized, “second” or slow pain (conducting signals at a rate of less than 1.0 m/s). Neuroanatomical and molecular characterization of nociceptors has further demonstrated their heterogeneity, particularly for the C fibers (Basbaum et al., 2009). For example, the so-called “peptidergic” population of C nociceptors releases neuropeptides, substance P (SP), and calcitonin-gene related peptide (CGRP); they also 3 Introduction express the TrkA neurotrophin receptor, which responds to nerve growth factor (NGF). The nonpeptidergic population of C nociceptors expresses the c-Ret neurotrophin receptor that is targeted by glial-derived neurotrophic factor (GDNF), and a large percentage of the c-Ret-positive population also binds the IB4 isolectin and expresses the specific purinergic P2X3 receptor subtypes (see also Paragraph 1.4.3, Ruan & Burnstock, 2003; Basbaum et al., 2009).