Medial prefrontal cortex diclofenac-induced antinociception is mediated through GPR55, cannabinoid CB1, and mu-opioid receptors of this area and periaqueductal gray

  • Esmaeal Tamaddonfard
  • Amir ErfanparastEmail author
  • Reza Salighedar
  • Sina Tamaddonfard
Original Article


Supraspinal mechanisms of non-steroidal anti-inflammatory drug (NSAID)-induced antinociception are not well understood. In the present study, the possible antinociceptive mechanisms induced by intra-medial prefrontal cortex (intra-mPFC) microinjection of diclofenac were investigated after blockade of GPR55, cannabinoid CB1, and mu-opioid receptors in this area and ventrolateral periaqueductal gray (vlPAG). For drug delivery, unilateral (left side) of mPFC and bilateral (right and left sides) of vlPAG were surgically cannulated. Formalin test was induced by subcutaneous injection of a diluted formalin solution into the right vibrissa pad. A typical biphasic (neurogenic and inflammatory phases) pain behavior was produced following formalin injection. Microinjection of diclofenac (2.5, 5, and 10 μg/0.25 μL) into the mPFC suppressed both phases of pain. Intra-mPFC microinjection of naloxonazine (a mu-opioid receptor antagonist, 1 μg/0.25 μL) and AM251 (a cannabinoid CB1 receptor antagonist, 1 μg/0.25 μL) increased both phases of pain intensity. In addition, intra-mPFC-microinjected diclofenac-induced antinociception was inhibited by prior intra-mPFC and intra-vlPAG administration of naloxonazine and AM251. On the other hand, intra-mPFC and intra-vlPAG microinjection of AM251 (0.25 μg/0.25 μL) decreased pain severity which was inhibited by prior administration of ML193. The above-mentioned drugs did not alter locomotor activity. In conclusion, diclofenac suppressed both the neurogenic and inflammatory phases of formalin-induced orofacial pain at the level of mPFC. GPR55, cannabinoid CB1, and mu-opioid receptors of the mPFC and vlPAG might be involved in the mPFC analgesic effects of diclofenac.


Diclofenac Cannabinoid receptors Opioid receptors Orofacial pain mPFC vlPAG 


Authors’ contributions

ET and AE conceived and designed the research. The experiments were conducted by RS and ST. ET and AE analyzed the data and wrote the manuscript. All of the authors read and approved the manuscript.

Funding information

This study was financially supported by the Faculty of Veterinary Medicine of Urmia University (grant no. 1396-03-22/D10-485).

Compliance with ethical standards

Ethical approval

All experiments were approved by the Veterinary Ethics Committee of the Faculty of Veterinary Medicine of Urmia University.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Ahmad T, Lauzon NM, de Jaeger X, Laviolette SR (2013) Cannabinoid transmission in the prelimbic cortex bidirectionally controls opiate reward and aversion signaling through dissociable kappa versus μ-opiate receptor dependent mechanisms. J Neurosci 33:15642–15651PubMedPubMedCentralCrossRefGoogle Scholar
  2. Akhtar F, Haque T, Sato F, Kato T, Ohara H, Fujio T, Tsutsumi K, Uchino K, Sessle BJ, Yoshida A (2014) Projections from the dorsal peduncular cortex to the trigeminal subnucleus caudalis (medullary dorsal horn) and other lower brainstem areas in rats. Neuroscience 266:23–37CrossRefGoogle Scholar
  3. Alexander SP, Christopoulos A, Davenport AP, Kelly E, Marrion NV, Peters JA, Faccenda E, Harding SD, Pawson AJ, Sharman JL, Southan C, Davies JA (2017) The concise guide to pharmacology 2017/18: G protein-coupled receptors. Br J Pharmacol 174:S17–S129PubMedPubMedCentralCrossRefGoogle Scholar
  4. Atzeni F, Masala IF, Sarzi-Puttini P (2018) A review of chronic musculoskeletal pain: central and peripheral effects of diclofenac. Pain Ther 7:163–177PubMedPubMedCentralCrossRefGoogle Scholar
  5. Bialuk I, Winnicka MM (2011) AM251, cannabinoids receptors ligand, improves recognition memory in rats. Pharmacol Rep 63:670–679PubMedCrossRefPubMedCentralGoogle Scholar
  6. Borsook D, Burstein R, Becerra L (2004) Functional imaging of the human trigeminal system: opportunities for new insights into pain processing in health and disease. J Neurobiol 61:107–125PubMedCrossRefPubMedCentralGoogle Scholar
  7. Burgos E, Pascual D, Martín MI, Goicoechea C (2010) Antinociceptive effect of the cannabinoid agonist, WIN 55,212-2, in the orofacial and temporomandibular formalin tests. Eur J Pain 14:40–48PubMedCrossRefPubMedCentralGoogle Scholar
  8. Carey LM, Gutierrez T, Deng L, Lee WH, Mackie K, Hohmann AG (2017) Inflammatory and neuropathic nociception is preserved in GPR55 knockout mice. Sci Rep 7:944PubMedPubMedCentralCrossRefGoogle Scholar
  9. Carlsson KH, Helmreich J, Jurna I (1986) Activation of inhibition from the periaqueductal grey matter mediates central analgesic effect of metamizol (dipyrone). Pain 27:373–390PubMedCrossRefPubMedCentralGoogle Scholar
  10. Cheriyan J, Sheets PL (2018) Altered excitability and local connectivity of mPFC-PAG neurons in a mouse model of neuropathic pain. J Neurosci 38:4829–4839PubMedPubMedCentralCrossRefGoogle Scholar
  11. Cheriyan J, Kaushik MK, Ferreira AN, Sheets PL (2016) Specific targeting of the basolateral amygdala to projectionally defined pyramidal neurons in prelimbic and infralimbic cortex. eNeuro. PubMedPubMedCentralCrossRefGoogle Scholar
  12. Chichorro JG, Lorenzetti BB, Zampronio AR (2004) Involvement of bradykinin, cytokines, sympathetic amines and prostaglandins in formalin-induced orofacial nociception in rats. Br J Pharmacol 141:1175–1184PubMedPubMedCentralCrossRefGoogle Scholar
  13. Clavelou P, Dallel R, Orliaguet T, Woda A, Raboisson P (1995) Theorofacial formalin test in rats: effects of different formalin concentrations. Pain 62:295–301PubMedCrossRefPubMedCentralGoogle Scholar
  14. DaSilva AF, Nascimento TD, DosSantos MF, Lucas S, van Holsbeeck H, DeBoer M, Maslowski E, Love T, Martikainen IK, Koeppe RA, Smith YR, Zubieta JK (2014) Association of μ-opioid activation in the prefrontal cortex with spontaneous migraine attacks - brief report I. Ann Clin Transl Neurol 1:439–444PubMedPubMedCentralCrossRefGoogle Scholar
  15. Der-Ghazarian T, Widarma CB, Gutierrez A, Amodeo LR, Valentine JM, Humphrey DE, Gonzalez AE, Crawford CA, McDougall SA (2014) Behavioral effects of dopamine receptor inactivation in the caudate-putamen of preweanling rats: role of the D2 receptor. Psychopharmacology 231:651–662PubMedCrossRefPubMedCentralGoogle Scholar
  16. Erfanparast A, Tamaddonfard E, Taati M, Dabaghi M (2015) Role of the thalamic submedius nucleus histamine H1 and H2 and opioid receptors in modulation of formalin-induced orofacial pain in rats. Naunyn-Schmiedeberg's Arc Pharmacol 388(10):1089–1096CrossRefGoogle Scholar
  17. Erfanparast A, Tamaddonfard E, Nemati S (2017) Effects of intra-hippocampal microinjection of vitamin B12 on the orofacial pain and memory impairments induced by scopolamine and orofacial pain in rats. Physiol Behav 170:68–77PubMedCrossRefPubMedCentralGoogle Scholar
  18. Escobar W, Ramirez K, Avila C, Limongi R, Vanegas H, Vazquez E (2012) Metamizol, a non-opioid analgesic, acts via endocannabinoids in the PAG-RVM axis during inflammation in rats. Eur J Pain 16:676–689PubMedCrossRefPubMedCentralGoogle Scholar
  19. Floyd NS, Price JL, Ferry AT, Keay KA, Bandler R (2000) Orbitomedial prefrontal cortical projections to distinct longitudinal columns of the periaqueductal gray in the rat. J Comp Neurol 422:556–578PubMedCrossRefPubMedCentralGoogle Scholar
  20. Fossum EN, Lisowski MJ, Macey TA, Ingram SL, Morgan MM (2008) Microinjection of the vehicle dimethyl sulfoxide (DMSO) into the periaqueductal gray modulates morphine antinociception. Brain Res 1204:53–58PubMedCrossRefPubMedCentralGoogle Scholar
  21. Freitas RL, Salgado-Rohner CJ, Hallak JE, Crippa JA, Coimbra NC (2013) Involvement of prelimbic medial prefrontal cortex in panic-like elaborated defensive behaviour and innate fear-induced antinociception elicited by GABAA receptor blockade in the dorsomedial and ventromedial hypothalamic nuclei: role of the endocannabinoid CB1 receptor. Int J Neuropsychopharmacol 16:1781–1798PubMedCrossRefPubMedCentralGoogle Scholar
  22. Gabbott PL, Warner TA, Jays PR, Salway P, Busby SJ (2005) Prefrontal cortex in the rat: projections to subcortical autonomic, motor, and limbic centers. J Comp Neurol 492:145–177PubMedCrossRefPubMedCentralGoogle Scholar
  23. Grim TW, Ghosh S, Hsu KL, Cravatt BF, Kinsey SG, Lichtman AH (2014) Combined inhibition of FAAH and COX produces enhanced anti-allodynic effects in mouse neuropathic and inflammatory pain models. Pharmacol Biochem Behav 124:405–411PubMedPubMedCentralCrossRefGoogle Scholar
  24. Guerrero-Alba R, Barragán-Iglesias P, González-Hernández A, Valdez-Moráles EE, Granados-Soto V, Condés-Lara M, Rodríguez MG, Marichal-Cancino BA (2018) Some prospective alternatives for treating pain: the endocannabinoid system and its putative receptors GPR18 and GPR55. Front Pharmacol 9:1496PubMedCrossRefPubMedCentralGoogle Scholar
  25. Gurtskaia G, Tsiklauri N, Nozadze I, Nebieridze M, Tsagareli MG (2014) Antinociceptive tolerance to NSAIDs microinjected into dorsal hippocampus. BMC Pharmacol Toxicol 15:10PubMedPubMedCentralCrossRefGoogle Scholar
  26. Gwanyanya A, Macianskiene R, Mubagwa KJ (2012) Insights into the effects of diclofenac and other non-steroidal anti-inflammatory agents on ion channels. Pharm Pharmacol 64:1359–1375CrossRefGoogle Scholar
  27. Henstridge CM, Balenga NA, Schröder R, Kargl JK, Platzer W, Martini L, Arthur S, Penman J, Whistler JL, Kostenis E, Waldhoer M (2010) GPR55 ligands promote receptor coupling to multiple signalling pathways. Br J Pharmacol 160:604–614PubMedPubMedCentralCrossRefGoogle Scholar
  28. Hesselink JMK, Hekke TA (2012) Therapeutic utility of palmitoylethanolamide in the treatment of neuropathic pain associated with various pathological conditions: a case series. J Pain Res 5:437–442PubMedPubMedCentralCrossRefGoogle Scholar
  29. Johnson SB, Emmons EB, Lingg RT, Anderson RM, Romig-Martin SA, LaLumiere RT, Narayanan NS, Viau V, Radley JJ (2018) Prefrontal-bed nucleus circuit modulation of a passive coping response set. J Neurosci 39: 1421-1418PubMedCrossRefPubMedCentralGoogle Scholar
  30. Kalyuzhny AE, Arvidsson U, Wu W, Wessendorf MW (1996) mu-Opioid and delta-opioid receptors are expressed in brainstem antinociceptive circuits: studies using immunocytochemistry and retrograde tract-tracing. J Neurosci 16:6490–6503PubMedCrossRefPubMedCentralGoogle Scholar
  31. Kaplan AA, Yurt KK, Deniz ÖG, Altun G (2018) Peripheral nerve and diclofenac sodium: molecular and clinical approaches. J Chem Neuroanat 87:2–11PubMedCrossRefPubMedCentralGoogle Scholar
  32. Kiritoshi T, Ji G, Neugebauer V (2016) Rescue of impaired mGluR5-driven endocannabinoid signaling restores prefrontal cortical output to inhibit pain in arthritic rats. J Neurosc 36: 837-850PubMedPubMedCentralCrossRefGoogle Scholar
  33. Lee SW, Stanley BG (2005) NMDA receptors mediate feeding elicited by neuropeptide Y in the lateral and perifornical hypothalamus. Brain Res 1063:1–8PubMedCrossRefPubMedCentralGoogle Scholar
  34. Lee HJ, Chang LY, Ho YC, Teng SF, Hwang LL, Mackie K, Chiou LC (2016) Stress induces analgesia via orexin1 receptor-initiated endocannabinoid/CB1 signaling in the mouse periaqueductal gray. Neuropharmacology 105:577–586PubMedCrossRefPubMedCentralGoogle Scholar
  35. Li YQ, Shinonaga Y, Takada M, Mizuno N (1993a) Demonstration of axon terminals of projection fibers from the periaqueductal gray onto neurons in the nucleus raphe magnus which send their axons to the trigeminal sensory nuclei. Brain Res 608:138–140PubMedCrossRefPubMedCentralGoogle Scholar
  36. Li YQ, Takada M, Shinonaga Y, Mizuno N (1993b) Direct projections from the midbrain periaqueductal gray and the dorsal raphe nucleus to the trigeminal sensory complex in the rat. Neuroscience 54:431–443PubMedCrossRefPubMedCentralGoogle Scholar
  37. Mansour A, Fox CA, Akil H, Watson SJ (1995) Opioid-receptor mRNA expression in the rat CNS: anatomical and functional implications. Trends Neurosci 18:22–29PubMedCrossRefPubMedCentralGoogle Scholar
  38. Marichal-Cancino BA, Fajardo-Valdez A, Ruiz-Contreras AE, Mendez-Díaz M, Prospero-García O (2017) Advances in the physiology of GPR55 in the central nervous system. Curr Neuropharmacol 15:771–778PubMedCrossRefPubMedCentralGoogle Scholar
  39. Matheus MG, de-Lacerda JC, Guimarães FS (1997) Behavioral effects of “vehicle” microinjected into the dorsal periaqueductal grey of rats tested in the elevated plus maze. Braz J Med Biol Res 30:61–64PubMedCrossRefPubMedCentralGoogle Scholar
  40. Miranda HF, Sierralta F, Lux S, Troncoso R, Ciudad N, Zepeda R, Zanetta P, Noriega V, Prieto JC (2015) Involvement of nitridergic and opioidergic pathways in the antinociception of gabapentin in the orofacial formalin test in mice. Pharmacol Rep 67:399–403PubMedCrossRefPubMedCentralGoogle Scholar
  41. Naderi N, Majidi M, Mousavi Z, Khoramian Tusi S, Mansouri Z, Khodagholi F (2012) The interaction between intrathecal administration of low doses of palmitoylethanolamide and AM251 in formalin-induced pain related behavior and spinal cord IL1-β expression in rats. Neurochem Res 37:778–785PubMedCrossRefPubMedCentralGoogle Scholar
  42. Ong WY, Stohler CS, Herr DR (2019) Role of the prefrontal cortex in pain processing. Mol Neurobiol 56:1137–1166PubMedCrossRefPubMedCentralGoogle Scholar
  43. Ossipov MH (2012) The perception and endogenous pain modulation. Scientifica (Cairo) 2012:561761Google Scholar
  44. Patrick GW, Robinson MA (1987) Collateral projection from trigeminal sensory nuclei to ventrobasal thalamus and cerebellar cortex. J Comp Neurol 192:229–236Google Scholar
  45. Paxinos G, Watson C (2007) The rat brain in stereotaxic coordinates, 6th edn. Elsevier, New YorkGoogle Scholar
  46. Pereira-Leite C, Nunes C, Jamal SK, Cuccovia IM, Reis S (2017) Nonsteroidal anti-inflammatory therapy: a journey toward safety. Med Res Rev 37:802–859PubMedCrossRefPubMedCentralGoogle Scholar
  47. Pirkulashvili N, Tsiklauri N, Nebieridze M, Tsagareli MG (2017) Antinociceptive tolerance to NSAIDs in the agranular insular cortex is mediated by opioid mechanism. J Pain Res 10:1561PubMedPubMedCentralCrossRefGoogle Scholar
  48. Ramanathan KR, Jin J, Giustino TF, Payne MR, Maren S (2018) Prefrontal projections to the thalamic nucleus reuniens mediate fear extinction. Nat Commun 9:4527PubMedPubMedCentralCrossRefGoogle Scholar
  49. Rea K, McGowan F, Corcoran L, Roche M, Finn DP (2018) The prefrontal cortical endocannabinoid system modulates fear-pain interactions in a subregion-specific manner. Br J Pharmacol 176:1492–1505PubMedCrossRefPubMedCentralGoogle Scholar
  50. Ryberg E, Larsson N, Sjogren S, Hjorth S, Hermansson NO, Leonova J, Elebring T, Nilsson K, Drmota T, Greasley PJ (2007) The orphan receptor GPR55 is a novel cannabinoid receptor. Br J Pharmacol 152:1092–1101PubMedPubMedCentralCrossRefGoogle Scholar
  51. Sandri A (2016) Spinal antinflammatory action of diclofenac. Minerva Med 107:167–172PubMedPubMedCentralGoogle Scholar
  52. Selleck RA, Baldo BA (2017) Feeding-modulatory effects of mu-opioids in the medial prefrontal cortex: a review of recent findings and comparison to opioid actions in the nucleus accumbens. Psychopharmacology 234:1439–1449PubMedPubMedCentralCrossRefGoogle Scholar
  53. Sessle BJ (2011) Peripheral and central mechanisms of orofacial inflammatory pain. Int Rev Neurobiol 97:179–206PubMedCrossRefPubMedCentralGoogle Scholar
  54. Silva LC, Miranda e Castor MG, Souza TC, Duarte ID, Romero TR (2015) NSAIDs induce peripheral antinociception by interaction with the adrenergic system. Life Sci 130:7–11PubMedCrossRefPubMedCentralGoogle Scholar
  55. Silva LC, Castor MG, Navarro LC, Romero TR, Duarte ID (2016) κ-Opioid receptor participates of NSAIDs peripheral antinociception. Neurosci Lett 622:6–9PubMedCrossRefPubMedCentralGoogle Scholar
  56. Taati M, Tamaddonfard E (2018) Ventrolateral orbital cortex oxytocin attenuates neuropathic pain through periaqueductal gray opioid receptor. Pharmacol Rep 70:577–583PubMedCrossRefPubMedCentralGoogle Scholar
  57. Tamaddonfard E, Tamaddonfard S, Cheraghiyan S (2018) Effects of intracerebroventricular injection of vitamin B12 on formalin-induced muscle pain in rats: role of cyclooxygenase pathway and opioid receptors. Vet Res Forum 9:329–335PubMedPubMedCentralGoogle Scholar
  58. Tortorici V, Vanegas H (2000) Opioid tolerance induced by metamizol (dipyrone) microinjections into the periaqueductal gray of rats. Eur J Neurosci 12:4074–4080PubMedCrossRefPubMedCentralGoogle Scholar
  59. Tortorici V, Nogueira L, Aponte Y, Vanegas H (2004) Involvement of cholecystokinin in the opioid tolerance induced by dipyrone (metamizol) microinjections into the periaqueductal gray matter of rats. Pain 112:113–120PubMedCrossRefPubMedCentralGoogle Scholar
  60. Tsiklauri N, Pirkulashvili N, Nozadze I, Nebieridze M, Gurtskaia G, Abzianidze E, Tsagareli MG (2018) Antinociceptive tolerance to NSAIDs in the anterior cingulate cortex is mediated via endogenous opioid mechanism. BMC Pharmacol Toxicol 19:2PubMedPubMedCentralCrossRefGoogle Scholar
  61. Tsou K, Brown S, Sañudo-Peña MC, Mackie K, Walker JM (1998) Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system. Neuroscience 83:393–411PubMedCrossRefPubMedCentralGoogle Scholar
  62. Van der Cruyssen F, Politis C (2018) Neurophysiological aspects of the trigeminal sensory system: an update. Rev Neurosci 29:115–123PubMedCrossRefPubMedCentralGoogle Scholar
  63. Vanegas H, Tortorici V (2002) Opioidergic effects of nonopioid analgesics on the central nervous system. Cell Mol Neurobiol 22:655–661PubMedCrossRefPubMedCentralGoogle Scholar
  64. Vanegas H, Vazquez E, Tortorici V (2010) NSAIDs, opioids, cannabinoids and the control of pain by the central nervous system. Pharmaceuticals (Basel) 3:1335–1347CrossRefGoogle Scholar
  65. Vazquez E, Hernandez N, Escobar W, Vanegas H (2005) Antinociception induced by intravenous dipyrone (metamizol) upon dorsal horn neurons: involvement of endogenous opioids at the periaqueductal gray matter, the nucleus raphe magnus, and the spinal cord in rats. Brain Res 1048:211–217PubMedCrossRefPubMedCentralGoogle Scholar
  66. Vučković S, Srebro D, Vujović KS, Vučetić Č, Prostran M (2018) Cannabinoids and pain: new insights from old molecules. Front Pharmacol 9:1259PubMedPubMedCentralCrossRefGoogle Scholar
  67. Vuilleumier PH, Schliessbach J, Curatolo M (2018) Current evidence for central analgesic effects of NSAIDs: an overview of the literature. Minerva Anestesiol 84:865–870PubMedPubMedCentralGoogle Scholar
  68. Ward SJ, Raffa RB (2011) Rimonabant redux and strategies to improve the future outlook of CB1 receptor neutral-antagonist/inverse-agonist therapies. Obesity (Silver Spring) 19:1325–1334CrossRefGoogle Scholar
  69. Wilson-Poe AR, Morgan MM, Aicher SA, Hegarty DM (2012) Distribution of CB1 cannabinoid receptors and their relationship with mu-opioid receptors in the rat periaqueductal gray. Neuroscience 213:191–200PubMedPubMedCentralCrossRefGoogle Scholar
  70. Woodhams SG, Chapman V, Finn DP, Hohmann AG, Neugebauer V (2017) The cannabinoid system and pain. Neuropharmacology 124:105–120PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Esmaeal Tamaddonfard
    • 1
  • Amir Erfanparast
    • 1
    Email author
  • Reza Salighedar
    • 1
  • Sina Tamaddonfard
    • 1
  1. 1.Division of Physiology, Department of Basic Sciences, Faculty of Veterinary MedicineUrmia UniversityUrmiaIran

Personalised recommendations