Purinergic neuron/glia communication in mouse trigeminal ganglia and its crosstalk with known algogenic mediators: implications for basic mechanisms of migraine pain

Aims: to study the role of the purinergic system in the neuron-to-glial cell communication within the trigeminal ganglion, and its cross-talk with known pro-algogenic systems (such as bradykinin, BK, and calcitonin gene-related peptide, CGRP). The final goal is the identification of new cellular and molecular players in the onset and maintenance of trigeminal-associated pain, for the development of new effective therapeutic strategies for migraine. Methods: primary trigeminal mixed neuron-glia or purified glial cultures were prepared from both wild type (WT) and transgenic R192Q CACNA1A knock-in (KI) mice, an animal model of familial hemiplegic migraine type 1 (FHM1). G protein-coupled P2Y receptor function was evaluated by single cell calcium imaging, and the extracellular concentrations of CGRP were measured by an ELISA assay. Immunocytochemical and immunohistochemical analyses were also performed. Results: a prolonged exposure of mixed neuron-glia cultures to BK significantly increases P2Y receptor-mediated calcium responses in glial cells, due to the BKinduced neuronal release of CGRP which in turn activates the ERK1/2 MAPkinase pathway on glial cells leading to P2Y receptor potentiation. Exposure of purified glial cultures to CGRP also leads to a significant release of pro- and anti-inflammatory cytokines, suggesting the generation of a complex inflammatory milieu. Interestingly, both basal and BK-stimulated CGRP release was higher in cultures from CACNA1A KI mice, suggesting a higher sensitivity of these cultures to pro-algogenic stimuli. Indeed, BK significantly up-regulated the number of glial cells showing functional P2Y receptors, particularly the UTP-sensitive subtypes, only in cultures from KI and not from WT mice. Conclusions: here we show, for the first time, that P2Y receptors on glial cells act as novel players in the cell-to-cell communication in the trigeminal ganglion underlying migraine pathophysiology and might therefore represent new targets for the development of innovative therapeutic agents against migraine pain.