PURINERGIC TRANSMISSION IN MIGRAINE: ROLE OF P2Y RECEPTORS IN THE SPINAL-TRIGEMINAL SYSTEM IN VIVO AND IN VITRO

Migraine is a highly disabling neurovascular disorder, affecting about 15% of adults in the Western World, with considerable socio-economic implications on both productivity and quality of life (Goadsby, 2007). Despite the progresses made in migraine research during the past few decades, there is little or no evidence that shows with certainty which and how pain fibers are activated during migraine attacks. Most investigators currently believe that a migraine attack starts in the brain, as suggested by premonitory symptoms. However, a debate about migraine origin is still ongoing: some scientists argue for a central origin of the headache pain (Goadsby et al., 2009), whereas others, including our research group, believe that the headache is triggered by activation of peripheral nociceptors. Indeed, the trigeminal nerve conveys sensory information from most extracranial and intracranial structures to the spinal trigeminal nucleus, and it has been therefore hypothesized that migraine could arise from a primary dysfunction leading to activation and sensitization of the trigemino-vascular system (Olesen et al., 2009). On this basis, the final aim of my study have been setting up both in vitro and in vivo models, as tools for evaluating the molecular and cellular role of the purinergic system in trigeminal pain transmission, in order to identify new potential targets for migraine pain therapy. In the first part of my PhD experimental work we have characterized the presence, functionality, and cellular localization of P2 receptors in primary mixed neuron-glia cultures prepared from mouse TG. Our results show that TG sensory neurons mainly express functional P2X3 receptors, while SGCs are highly responsive to P2Y-agonists (i.e., ADP, UTP, and UDP). Moreover, exposure to the pro-inflammatory agent BK significantly modified P2X3 receptor function in a biphasic way, with an upregulation or a reduction of alpha,beta-meATP-mediated calcium responses after an acute or chronic exposure, respectively. We also demonstrated that chronic exposure to the pro-inflammatory agent BK significantly enhanced the functionality of P2Y-receptor expressed by SGCs. Our attempt to dissect the molecular pathways associated to this effect, has led to the discovery of a complex neuron to glia signaling. Indeed, we demonstrate that CGRP released from sensory neurons is the key mediator of BK-induced P2Y receptor upregulation on SCGs, and that the latter occurs via the ERK1/2 MAP kinase pathway. In addition, we investigated both CGRP release and changes in P2Y receptor functionalities in a genetic mouse model of migraine in which the R192Q missense mutation, that causes familial hemiplegic migraine type 1 (FHM1), was introduced in the α1 subunit of CaV2.1 calcium channels by a gene targeting approach (van den Maagdenberg et al., 2004). While there was no difference in BK-induced P2Y receptor upregulation in SCGs, between from wild type and CaV2.1 α1 R192Q mutant KI cultures, a significant increase in the number of SGCs responding both to ADP and UTP was selectively found in KI cultures treated with BK. In the last part of my PhD project we set up in vivo models of trigeminal pain. By using a model of acute pain we have demonstrated that the specific knock down of P2Y4 receptor expression in vivo did not produce substantial changes in pain behavior following intra-lip injection of formalin. We have also set up a model of sub-chronic inflammation, based on injection of CFA into the rat TMJ, and we have characterized the activation glial cells populations in both TG and brainstem. Specifically, we demonstrated CFA injection is associated with activation of SGCs and macrophages in the TG, as well as of microglial cells in the spinal trigeminal nucleus, with no signs of reactive astrogliosis. We also demonstrated that the expression levels of the microglial P2Y12 receptor do not change following CFA injections. In conclusion, this in vivo model represents a useful tool for evaluating the role of specific P2Y receptor subtypes, expressed by glial cells, in the development and maintenance of chronic trigeminal pain and migraine-associated pain. We intend now to look for possible changes in other P2Y receptors expressed by glial cells, and to evaluate the pro- or anti-algogenic effects of their pharmacological or biotechnological inhibition.