Journal of Neuroimmune Pharmacology

, Volume 9, Issue 5, pp 668–678 | Cite as

P2X4 Receptor Regulates Alcohol-Induced Responses in Microglia

  • Larisa Gofman
  • Jonathan M. Cenna
  • Raghava Potula


Mounting evidence indicates that alcohol-induced neuropathology may result from multicellular responses in which microglia cells play a prominent role. Purinergic receptor signaling plays a key role in regulating microglial function and, more importantly, mediates alcohol-induced effects. Our findings demonstrate that alcohol increases expression of P2X4 receptor (P2X4R), which alters the function of microglia, including calcium mobilization, migration and phagocytosis. Our results show a significant up-regulation of P2X4 gene expression as analyzed by real-time qPCR (***p < 0.002) and protein expression as analyzed by flow cytometry (**p < 0.004) in embryonic stem cell-derived microglial cells (ESdM) after 48 hours of alcohol treatment, as compared to untreated controls. Calcium mobilization in ethanol treated ESdM cells was found to be P2X4R dependent using 5-BDBD, a P2X4R selective antagonist. Alcohol decreased migration of microglia towards fractalkine (CX3CL1) by 75 % following 48 h of treatment compared to control (***p < 0.001). CX3CL1-dependent migration was confirmed to be P2X4 receptor-dependent using the antagonist 5-BDBD, which reversed the effects as compared to alcohol alone (***p < 0.001). Similarly, 48 h of alcohol treatment significantly decreased phagocytosis of microglia by 15 % compared to control (*p < 0.05). 5-BDBD pre-treatment prior to alcohol treatment significantly increased microglial phagocytosis (***p < 0.001). Blocking P2X4R signaling with 5-BDBD decreased the level of calcium mobilization compared to ethanol treatment alone. These findings demonstrate that P2X4 receptor may play a role in modulating microglial function in the context of alcohol abuse.


Microglia Alcohol Purinergic receptor X4 



The authors would like to acknowledge Nancy L. Reichenbach for editing and Dr. Uma Sriram for critical reading of the manuscript, and Dr. Slava Rom for his expert help with the graphics.

Supplementary material

11481_2014_9559_MOESM1_ESM.docx (8.7 mb)
ESM 1 (DOCX 8875 kb)


  1. Aroor AR, Baker RC (1998) Ethanol inhibition of phagocytosis and superoxide anion production by microglia. Alcohol 15:277–280PubMedCrossRefGoogle Scholar
  2. Asatryan L, Popova M, Perkins D, Trudell JR, Alkana RL, Davies DL (2010) Ivermectin antagonizes ethanol inhibition in purinergic P2X4 receptors. J Pharmacol Exp Ther 334:720–728PubMedCrossRefPubMedCentralGoogle Scholar
  3. Asatryan L, Nam HW, Lee MR, Thakkar MM, Saeed Dar M, Davies DL, Choi DS (2011) Implication of the purinergic system in alcohol use disorders. Alcohol-Clin Exp Res 35:584–594PubMedCrossRefPubMedCentralGoogle Scholar
  4. Asatryan L, Yardley MM, Khoja S, Trudell JR, Hyunh N, Louie SG, Petasis NA, Alkana RL, Davies DL (2014) Avermectins differentially affect ethanol intake and receptor function: implications for developing new therapeutics for alcohol use disorders. Int J Neuropsychopharmacol 1–10Google Scholar
  5. Beutner C, Roy K, Linnartz B, Napoli I, Neumann H (2010) Generation of microglial cells from mouse embryonic stem cells. Nat Protoc 5:1481–1494PubMedCrossRefGoogle Scholar
  6. Boucsein C, Zacharias R, Farber K, Pavlovic S, Hanisch UK, Kettenmann H (2003) Purinergic receptors on microglial cells: functional expression in acute brain slices and modulation of microglial activation in vitro. Eur J Neurosci 17:2267–2276PubMedCrossRefGoogle Scholar
  7. Boyadjieva NI, Sarkar DK (2010) Role of microglia in ethanol’s apoptotic action on hypothalamic neuronal cells in primary cultures. Alcohol-Clin Exp Res 34:1835–1842PubMedCrossRefPubMedCentralGoogle Scholar
  8. Burnstock G (2008) Purinergic signalling and disorders of the central nervous system. Nat Rev Drug Discov 7:575–590PubMedCrossRefGoogle Scholar
  9. Burnstock G (2013) Introduction to purinergic signalling in the brain. Adv Exp Med Biol 986:1–12PubMedCrossRefGoogle Scholar
  10. Burnstock G, Williams M (2000) P2 purinergic receptors: modulation of cell function and therapeutic potential. J Pharmacol Exp Ther 295:862–869PubMedGoogle Scholar
  11. Chimini G, Chavrier P (2000) Function of Rho family proteins in actin dynamics during phagocytosis and engulfment. Nat Cell Biol 2:E191–E196PubMedCrossRefGoogle Scholar
  12. Davies DL, Kochegarov AA, Kuo ST, Kulkarni AA, Woodward JJ, King BF, Alkana RL (2005) Ethanol differentially affects ATP-gated P2X(3) and P2X(4) receptor subtypes expressed in Xenopus oocytes. Neuropharmacology 49:243–253PubMedCrossRefGoogle Scholar
  13. Davies DL, Asatryan L, Kuo ST, Woodward JJ, King BF, Alkana RL, Xiao C, Ye JH, Sun H, Zhang L, Hu XQ, Hayrapetyan V, Lovinger DM, Machu TK (2006) Effects of ethanol on adenosine 5′-triphosphate-gated purinergic and 5-hydroxytryptamine receptors. Alcohol Clin Exp Res 30:349–358PubMedCrossRefPubMedCentralGoogle Scholar
  14. Deehan GA Jr, Brodie MS, Rodd ZA (2013) What is in that drink: the biological actions of ethanol, acetaldehyde, and salsolinol. Curr Top Behav Neurosci 13:163–184PubMedCrossRefGoogle Scholar
  15. den Hartog CR, Beckley JT, Smothers TC, Lench DH, Holseberg ZL, Fedarovich H, Gilstrap MJ, Homanics GE, Woodward JJ (2013) Alterations in ethanol-induced behaviors and consumption in knock-in mice expressing ethanol-resistant NMDA receptors. PLoS One 8:e80541CrossRefGoogle Scholar
  16. Dolganiuc A, Szabo G (2009) In vitro and in vivo models of acute alcohol exposure. World J Gastroenterol: WJG 15:1168–1177PubMedCrossRefPubMedCentralGoogle Scholar
  17. Dooley R, Mashukova A, Toetter B, Hatt H, Neuhaus EM (2011) Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium. BMC Neurosci 12:86PubMedCrossRefPubMedCentralGoogle Scholar
  18. Farber K, Kettenmann H (2006) Purinergic signaling and microglia. Pflugers Arch - Eur J Physiol 452:615–621CrossRefGoogle Scholar
  19. Fernandez-Lizarbe S, Pascual M, Guerri C (2009) Critical role of TLR4 response in the activation of microglia induced by ethanol. J Immunol 183:4733–4744PubMedCrossRefGoogle Scholar
  20. Glaser T, Resende RR, Ulrich H (2013) Implications of purinergic receptor-mediated intracellular calcium transients in neural differentiation. Cell Commun Signal: CCS 11:12PubMedCrossRefPubMedCentralGoogle Scholar
  21. Harper C (2009) The neuropathology of alcohol-related brain damage. Alcohol Alcohol 44:136–140PubMedCrossRefGoogle Scholar
  22. Hauser SR, Getachew B, Taylor RE, Tizabi Y (2011) Alcohol induced depressive-like behavior is associated with a reduction in hippocampal BDNF. Pharmacol Biochem Behav 100:253–258PubMedCrossRefPubMedCentralGoogle Scholar
  23. Inoue K (2006) The function of microglia through purinergic receptors: neuropathic pain and cytokine release. Pharmacol Ther 109:210–226PubMedCrossRefGoogle Scholar
  24. Inoue K (2008) Purinergic systems in microglia. Cell Mol Life Sci: CMLS 65:3074–3080PubMedCrossRefGoogle Scholar
  25. Inoue K, Tsuda M (2012a) Purinergic systems, neuropathic pain and the role of microglia. Exp Neurol 234:293–301PubMedCrossRefGoogle Scholar
  26. Inoue K, Tsuda M (2012b) P2X4 receptors of microglia in neuropathic pain. CNS & Neurol Disord Drug Targets 11:699–704CrossRefGoogle Scholar
  27. James G, Butt AM (2002) P2Y and P2X purinoceptor mediated Ca2+ signalling in glial cell pathology in the central nervous system. Eur J Pharmacol 447:247–260PubMedCrossRefGoogle Scholar
  28. Karavitis J, Murdoch EL, Deburghgraeve C, Ramirez L, Kovacs EJ (2012) Ethanol suppresses phagosomal adhesion maturation, Rac activation, and subsequent actin polymerization during FcgammaR-mediated phagocytosis. Cell Immunol 274:61–71PubMedCrossRefPubMedCentralGoogle Scholar
  29. Kettenmann H, Hanisch UK, Noda M, Verkhratsky A (2011) Physiology of microglia. Physiol Rev 91:461–553PubMedCrossRefGoogle Scholar
  30. Ko WH, Au CL, Yip CY (2003) Multiple purinergic receptors lead to intracellular calcium increases in cultured rat Sertoli cells. Life Sci 72:1519–1535PubMedCrossRefGoogle Scholar
  31. Kokoska ER, Smith GS, Deshpande Y, Wolff AB, Rieckenberg C, Miller TA (1999) Calcium accentuates injury induced by ethanol in human gastric cells. J gastrointest Surg: Official J Soc Surg Aliment Tract 3:308–318CrossRefGoogle Scholar
  32. Koshimizu TA, Van Goor F, Tomic M, Wong AO, Tanoue A, Tsujimoto G, Stojilkovic SS (2000) Characterization of calcium signaling by purinergic receptor-channels expressed in excitable cells. Mol Pharmacol 58:936–945PubMedGoogle Scholar
  33. Kosten TA (2011) Pharmacologically targeting the P2rx4 gene on maintenance and reinstatement of alcohol self-administration in rats. Pharmacol Biochem Behav 98:533–538PubMedCrossRefPubMedCentralGoogle Scholar
  34. Majumder P, Trujillo CA, Lopes CG, Resende RR, Gomes KN, Yuahasi KK, Britto LR, Ulrich H (2007) New insights into purinergic receptor signaling in neuronal differentiation, neuroprotection, and brain disorders. Purinergic Signal 3:317–331PubMedCrossRefPubMedCentralGoogle Scholar
  35. Marshall SA, McClain JA, Kelso ML, Hopkins DM, Pauly JR, Nixon K (2013) Microglial activation is not equivalent to neuroinflammation in alcohol-induced neurodegeneration: the importance of microglia phenotype. Neurobiol Dis 54:239–251PubMedCrossRefPubMedCentralGoogle Scholar
  36. McLarnon JG, Choi HB, Lue LF, Walker DG, Kim SU (2005) Perturbations in calcium-mediated signal transduction in microglia from Alzheimer’s disease patients. J Neurosci Res 81:426–435PubMedCrossRefGoogle Scholar
  37. Mei L, Du W, Gao W, Mei QB (2010) Purinergic signaling: a novel mechanism in immune surveillance. Acta Pharmacol Sin 31:1149–1153PubMedCrossRefPubMedCentralGoogle Scholar
  38. Moller T (2002) Calcium signaling in microglial cells. Glia 40:184–194PubMedCrossRefGoogle Scholar
  39. Napoli I, Kierdorf K, Neumann H (2009) Microglial precursors derived from mouse embryonic stem cells. Glia 57:1660–1671PubMedCrossRefGoogle Scholar
  40. Nelson S, Kolls JK (2002) Alcohol, host defence and society. Nat Rev Immunol 2:205–209PubMedCrossRefGoogle Scholar
  41. Neumann H, Kotter MR, Franklin RJ (2009) Debris clearance by microglia: an essential link between degeneration and regeneration. Brain: A J neurol 132:288–295CrossRefGoogle Scholar
  42. Nixon K, Kim DH, Potts EN, He J, Crews FT (2008) Distinct cell proliferation events during abstinence after alcohol dependence: microglia proliferation precedes neurogenesis. Neurobiol Dis 31:218–229PubMedCrossRefPubMedCentralGoogle Scholar
  43. Ohsawa K, Kohsaka S (2011) Dynamic motility of microglia: purinergic modulation of microglial movement in the normal and pathological brain. Glia 59:1793–1799PubMedCrossRefGoogle Scholar
  44. Ostrovskaya O, Asatryan L, Wyatt L, Popova M, Li K, Peoples RW, Alkana RL, Davies DL (2011) Ethanol is a fast channel inhibitor of P2X4 receptors. J Pharmacol Exp Ther 337:171–179PubMedCrossRefPubMedCentralGoogle Scholar
  45. Popova M, Trudell J, Li K, Alkana R, Davies D, Asatryan L (2013) Tryptophan 46 is a site for ethanol and ivermectin action in P2X4 receptors. Purinergic signalling 9(4):621–32PubMedCrossRefPubMedCentralGoogle Scholar
  46. Popp RL, Dertien JS (2008) Actin depolymerization contributes to ethanol inhibition of NMDA receptors in primary cultured cerebellar granule cells. Alcohol 42:525–539PubMedCrossRefPubMedCentralGoogle Scholar
  47. Potucek YD, Crain JM, Watters JJ (2006) Purinergic receptors modulate MAP kinases and transcription factors that control microglial inflammatory gene expression. Neurochem Int 49:204–214PubMedCrossRefGoogle Scholar
  48. Potula R, Hawkins BJ, Cenna JM, Fan S, Dykstra H, Ramirez SH, Morsey B, Brodie MR, Persidsky Y (2010) Methamphetamine causes mitrochondrial oxidative damage in human T lymphocytes leading to functional impairment. J Immunol 185:2867–2876PubMedCrossRefPubMedCentralGoogle Scholar
  49. Qureshi OS, Paramasivam A, Yu JC, Murrell-Lagnado RD (2007) Regulation of P2X4 receptors by lysosomal targeting, glycan protection and exocytosis. J Cell Sci 120:3838–3849PubMedCrossRefGoogle Scholar
  50. Ramirez SH, Fan S, Zhang M, Papugani A, Reichenbach N, Dykstra H, Mercer AJ, Tuma RF, Persidsky Y (2010a) Inhibition of glycogen synthase kinase 3beta (GSK3beta) decreases inflammatory responses in brain endothelial cells. Am J Pathol 176:881–892PubMedCrossRefPubMedCentralGoogle Scholar
  51. Ramirez SH, Fan SS, Dykstra H, Reichenbach N, Del Valle L, Potula R, Phipps RP, Maggirwar SB, Persidsky Y (2010b) Dyad of CD40/CD40 ligand fosters neuroinflammation at the blood–brain barrier and is regulated via JNK signaling: implications for HIV-1 encephalitis. J Neurosci 30:9454–9464PubMedCrossRefPubMedCentralGoogle Scholar
  52. Raouf R, Chabot-Dore AJ, Ase AR, Blais D, Seguela P (2007) Differential regulation of microglial P2X4 and P2X7 ATP receptors following LPS-induced activation. Neuropharmacology 53:496–504PubMedCrossRefGoogle Scholar
  53. Rimland D, Hand WL (1980) The effect of ethanol on adherence and phagocytosis by rabbit alveolar macrophages. J Lab Clin Med 95:918–926PubMedGoogle Scholar
  54. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675PubMedCrossRefGoogle Scholar
  55. Suk K (2007) Microglial signal transduction as a target of alcohol action in the brain. Curr Neurovasc Res 4:131–142PubMedCrossRefGoogle Scholar
  56. Szabo G (1997) Alcohol’s contribution to compromised immunity. Alcohol Health Res World 21:30–41PubMedGoogle Scholar
  57. Toulme E, Khakh BS (2012) Imaging P2X4 receptor lateral mobility in microglia: regulation by calcium and p38 MAPK. J biolo Chem 287:14734–14748CrossRefGoogle Scholar
  58. Trang T, Salter MW (2012) P2X4 purinoceptor signaling in chronic pain. Purinergic Signal 8:621–628PubMedCrossRefPubMedCentralGoogle Scholar
  59. Tsuda M, Tozaki-Saitoh H, Inoue K (2010) Pain and purinergic signaling. Brain Res Rev 63:222–232PubMedCrossRefGoogle Scholar
  60. Vazquez-Villoldo N, Domercq M, Martin A, Llop J, Gomez-Vallejo V, Matute C (2014) P2X4 receptors control the fate and survival of activated microglia. Glia 62:171–184PubMedCrossRefGoogle Scholar
  61. Verkhratsky A, Krishtal OA, Burnstock G (2009) Purinoceptors on neuroglia. Mol Neurobiol 39:190–208PubMedCrossRefGoogle Scholar
  62. Zhao YN, Wang F, Fan YX, Ping GF, Yang JY, Wu CF (2013) Activated microglia are implicated in cognitive deficits, neuronal death, and successful recovery following intermittent ethanol exposure. Behav Brain Res 236:270–282PubMedCrossRefGoogle Scholar
  63. Zou JY, Crews FT (2014) Release of neuronal HMGB1 by Ethanol through decreased HDAC activity activates brain neuroimmune signaling. PLoS One 9:e87915PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Larisa Gofman
    • 1
    • 2
  • Jonathan M. Cenna
    • 1
  • Raghava Potula
    • 1
    • 2
  1. 1.Department of Pathology and Laboratory MedicineTemple University School of MedicinePhiladelphiaUSA
  2. 2.Center for Substance Abuse Research, Temple University School of MedicinePhiladelphiaUSA

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