Research Paper Microglial P2X 4 R-evoked pain hypersensitivity is sexually dimorphic in rats Josiane C.S. Mapplebeck a,b,c , Rebecca Dalgarno d,e , YuShan Tu a , Orla Moriarty a,f , Simon Beggs a,g , Charlie H.T. Kwok d,e , Katherine Halievski a , Sofia Assi a,b , Jeffrey S. Mogil h,i , Tuan Trang d,e , Michael W. Salter a,b,c, * Abstract Microglia–neuron signalling in the spinal cord is a key mediator of mechanical allodynia caused by peripheral nerve injury. We recently reported sex differences in microglia in pain signalling in mice: spinal mechanisms underlying nerve injury–induced allodynia are microglial dependent in male but not female mice. Whether this sex difference in pain hypersensitivity mechanisms is conserved in other species is unknown. Here, we show that in rats, the spinal mechanisms of nerve injury–induced hypersensitivity in males differ from those in females, with microglial P2X 4 receptors (P2X 4 Rs) being a key point of divergence. In rats, nerve injury produced comparable allodynia and reactive microgliosis in both sexes. However, inhibiting microglia in the spinal cord reversed allodynia in male rats but not female rats. In addition, pharmacological blockade of P2X 4 Rs, by an intrathecally administered antagonist, attenuated pain hypersensitivity in male rats only. Consistent with the behavioural findings, nerve injury increased cell surface expression and function of P2X 4 Rs in acutely isolated spinal microglia from male rats but not from female rats. Moreover, in microglia cultured from male rats, but not in those from female rats, stimulating P2X 4 Rs drove intracellular signalling through p38 mitogen- activated protein kinase. Furthermore, chromatin immunoprecipitation–qPCR revealed that the transcription factor IRF5 differentially binds to the P2rx4 promoter region in female rats vs male rats. Finally, mechanical allodynia was produced in otherwise naive rats by intrathecally administering P2X 4 R-stimulated microglia from male rats but not those from female rats. Together, our findings demonstrate the existence of sexually dimorphic pain signalling in rats, suggesting that this sex difference is evolutionarily conserved, at least across rodent species. Keywords: Sex differences, Microglia, P2X 4 receptors, Nerve injury, Rats, Spinal cord, IRF5 1. Introduction Microglia-to-neuron signalling in the spinal cord dorsal horn is implicated in the initiation and maintenance of mechanical hypersensitivity (allodynia) after peripheral nerve injury (PNI). 2,8,14,41 Nerve injury induces microglial reactivity, which is characterized by cell proliferation, reconfiguration of the tran- scriptional network, changes in cell morphology, secretion of proinflammatory molecules, and upregulation of cell surface receptors. 1,40 Nerve injury increases expression of transcription factor IRF5, which drives microglial P2X 4 R upregulation in the spinal dorsal horn, a receptor necessary for the development of tactile allodynia after nerve injury. 19,21,41 Stimulating P2X 4 Rs, by ATP released from dorsal horn neurons, 20 leads to phosphory- lation and activation of p38-mitogen-activated protein kinase (p38-MAPK), and subsequently, drives synthesis and release of brain-derived neurotrophic factor (BDNF). 39 Secreted BDNF activates neuronal TrkB receptors resulting in reduced expres- sion of KCC2, a potassium chloride cotransporter, and ultimately, decreased efficacy of GABA A -mediated inhibition. 8,9,15 This disinhibition enhances nociceptive signalling to the brain and underlies the development of neuropathic pain. 16 However, the vast majority of literature implicating microglia– neuron signalling in pain, like the pain literature more generally, consists of experiments in which only male rodents were tested. 25,26 Failure to include female subjects is widespread and highlights a key gap in preclinical pain research, despite the predominance of chronic pain conditions in women. 3,12,24,26,38 Recent findings have revealed that the immune cells driving pain modulation are different between male and female mice. 33,34,36 In mice, we found that nerve injury induced similar levels of allodynia in both sexes. However, microglia are critical for spinal cord pain signalling only in males. 34 Although nerve injury produces equivalent spinal microglial reactivity in both sexes, pharmaco- logical or genetic interference with microglial functioning alle- viates allodynia in male mice only. 34 Specifically, inhibiting components of microglia–neuron signalling including P2X 4 Rs, Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article. J.C.S. Mapplebeck and R. Dalgarno are co-first authors. a Program in Neuroscience & Mental Health, Hospital for Sick Children, Toronto, ON, Canada, b Department of Physiology, University of Toronto, Toronto, ON, Canada, c University of Toronto Centre for the Study of Pain, Toronto, ON, Canada, d Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, AB, Canada, e Department of Physiology and Pharmacology, Hotchkiss Brain Institute, University of Calgary, Calgary, AB, Canada, f International Centre for Neurotherapeutics, Dublin City University, Dublin, Ireland, g UCL Great Ormond Street Institute of Child Health, London, United Kingdom, h Department of Psychology, McGill University, Montreal, QC, Canada, i Alan Edwards Centre for Research on Pain, McGill University, Montreal, QC, Canada *Corresponding author. Address: Hospital for Sick Children, 686 Bay St, Toronto, ON M5G 0A4, Canada. Tel.: (416) 813-6272; fax: (416) 813-7921. E-mail address: michael.salter@sickkids.ca (M.W. Salter). Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.painjournalonline.com). PAIN 159 (2018) 1752–1763 © 2018 International Association for the Study of Pain http://dx.doi.org/10.1097/j.pain.0000000000001265 1752 J.C.S. Mapplebeck et al. · 159 (2018) 1752–1763 PAIN ® Copyright Ó 2018 by the International Association for the Study of Pain. Unauthorized reproduction of this article is prohibited.