Korean J Physiol Pharmacol.  2019 Jul;23(4):271-279. 10.4196/kjpp.2019.23.4.271.

Potentiation of the glycine response by serotonin on the substantia gelatinosa neurons of the trigeminal subnucleus caudalis in mice

• Nguyen HT
• Cho DH
• Jang SH
• Han SK
• Park SJ

• KMID: 2450494
• DOI: http://doi.org/10.4196/kjpp.2019.23.4.271

Abstract

The lamina II, also called the substantia gelatinosa (SG), of the trigeminal subnucleus caudalis (Vc), is thought to play an essential role in the control of orofacial nociception. Glycine and serotonin (5-hydroxytryptamine, 5-HT) are the important neurotransmitters that have the individual parts on the modulation of nociceptive transmission. However, the electrophysiological effects of 5-HT on the glycine receptors on SG neurons of the Vc have not been well studied yet. For this reason, we applied the whole-cell patch clamp technique to explore the interaction of intracellular signal transduction between 5-HT and the glycine receptors on SG neurons of the Vc in mice. In nine of 13 neurons tested (69.2%), pretreatment with 5-HT potentiated glycine-induced current (I(Gly)). Firstly, we examined with a 5-HT₁ receptor agonist (8-OH-DPAT, 5-HT(1/7) agonist, co-applied with SB-269970, 5-HT₇ antagonist) and antagonist (WAY-100635), but 5-HT₁ receptor agonist did not increase IGly and in the presence of 5-HT₁ antagonist, the potentiation of 5-HT on I(Gly) still happened. However, an agonist (α-methyl-5-HT) and antagonist (ketanserin) of the 5-HT₂ receptor mimicked and inhibited the enhancing effect of 5-HT on I(Gly) in the SG neurons, respectively. We also verified the role of the 5-HT₇ receptor by using a 5-HT₇ antagonist (SB-269970) but it also did not block the enhancement of 5-HT on I(Gly). Our study demonstrated that 5-HT facilitated I(Gly) in the SG neurons of the Vc through the 5-HT₂ receptor. The interaction between 5-HT and glycine appears to have a significant role in modulating the transmission of the nociceptive pathway.

Keyword

Glycine receptor; Patch-clamp techniques; Serotonin; Substantia gelatinosa

MeSH Terms

Animals
Glycine*
Mice*
Neurons*
Neurotransmitter Agents
Nociception
Patch-Clamp Techniques
Receptors, Glycine
Serotonin*
Signal Transduction
Substantia Gelatinosa*
Glycine
Neurotransmitter Agents
Receptors, Glycine
Serotonin

Figure

• 

  Fig. 1 Potentiation of glycine-induced current (IGly) by 5-hydroxytryptamine (5-HT). (A) Representative trace shows the similarity of the repeated response induced by glycine (30 µM) and then the facilitating effect of 5-HT (10 µM) on IGly in substantia gelatinosa neuron. (B) Bar graph compares the average values obtained repeated application of glycine and glycine in the presence of 5-HT (*p < 0.05). NS, not significant.

• 

  Fig. 2 Actions of 5-HT1 agonist and antagonist on the 5-HT potentiation of glycine-induced current (IGly). (A) The represent trace shows that IGly was not increased in the presence of SB-269970 (10 µM), a 5-HT7 antagonist, and 8-OH-DPAT (30 µM), a 5-HT1/7 receptor agonist. (B) There was no significant difference between the mean relative amplitudes of IGly alone and IGly with 5-HT1 receptor agonist. (C) Glycine-induced inward current was increased by the simultaneous application of 5-HT (10 µM) and WAY-100635 (1 µM), a 5-HT1 selective antagonist. (D) There is a significant difference between the mean values produced by glycine alone and that produced by glycine in the presence of 5-HT and WAY-100635 (**p < 0.01). 5-HT, 5-hydroxytryptamine; NS, not significant.

• 

  Fig. 3 Actions of 5-HT2 agonist and antagonist on the 5-HT potentiation of glycine-induced current (IGly). (A) The represent trace showing IGly was potentiated by α-methyl 5-HT (30 µM), a 5-HT2 receptor-selective agonist. (B) Bar graph showing a statistical comparison of mean relative amplitudes between the application glycine (30 µM) alone and glycine in the presence of α-methyl 5-HT. (C) 5-HT (10 µM) failed to potentiate IGly in the presence of kentaserin (30 µM), a 5-HT2 receptor-selective antagonist. (D) Bar graph indicates no significant difference the mean relative amplitudes of IGly compared with IGly with 5-HT2 antagonist (*p < 0.05). 5-HT, 5-hydroxytryptamine; NS, not significant.

• 

  Fig. 4 Actions of 5-HT7 receptors on the 5-HT potentiation of glycine-induced current (IGly). (A) The represent trace illustrates that the IGly was still increased in the presence of both 5-HT (10 µM) and SB-269970 (10 µM), a 5-HT7 selective antagonist. (B) Bar graph demonstrates that there is a significant difference between the mean values created by glycine alone and by glycine in the simultaneous application of 5-HT and SB-269970 (*p < 0.01). 5-HT, 5-hydroxytryptamine.

Reference

1. Urban MO, Gebhart GF. Supraspinal contributions to hyperalgesia. Proc Natl Acad Sci U S A. 1999; 96:7687–7692.

2. Millan MJ. Descending control of pain. Prog Neurobiol. 2002; 66:355–474.

3. Suzuki R, Rygh LJ, Dickenson AH. Bad news from the brain: descending 5-HT pathways that control spinal pain processing. Trends Pharmacol Sci. 2004; 25:613–617.

4. Light AR, Casale EJ, Menétrey DM. The effects of focal stimulation in nucleus raphe magnus and periaqueductal gray on intracellularly recorded neurons in spinal laminae I and II. J Neurophysiol. 1986; 56:555–571.

5. Basbaum AI, Zahs K, Lord B, Lakos S. The fiber caliber of 5-HT immunoreactive axons in the dorsolateral funiculus of the spinal cord of the rat and cat. Somatosens Res. 1988; 5:177–185.

6. Light AR, Perl ER. Spinal termination of functionally identified primary afferent neurons with slowly conducting myelinated fibers. J Comp Neurol. 1979; 186:133–150.

7. Sugiura Y, Lee CL, Perl ER. Central projections of identified, unmyelinated (C) afferent fibers innervating mammalian skin. Science. 1986; 234:358–361.

8. Lorenzo LE, Ramien M, St Louis M, De Koninck Y, Ribeiro-da-Silva A. Postnatal changes in the Rexed lamination and markers of nociceptive afferents in the superficial dorsal horn of the rat. J Comp Neurol. 2008; 508:592–604.

9. Todd AJ. Anatomy of primary afferents and projection neurones in the rat spinal dorsal horn with particular emphasis on substance P and the neurokinin 1 receptor. Exp Physiol. 2002; 87:245–249.

10. Sessle BJ. Acute and chronic craniofacial pain: brainstem mechanisms of nociceptive transmission and neuroplasticity, and their clinical correlates. Crit Rev Oral Biol Med. 2000; 11:57–91.

11. Santos SF, Rebelo S, Derkach VA, Safronov BV. Excitatory interneurons dominate sensory processing in the spinal substantia gelatinosa of rat. J Physiol. 2007; 581(Pt 1):241–254.

12. Mitchell K, Spike RC, Todd AJ. An immunocytochemical study of glycine receptor and GABA in laminae I-III of rat spinal dorsal horn. J Neurosci. 1993; 13:2371–2381.

13. Todd AJ, Watt C, Spike RC, Sieghart W. Colocalization of GABA, glycine, and their receptors at synapses in the rat spinal cord. J Neurosci. 1996; 16:974–982.

14. Willis WD Jr. Coggeshall RE. Functional organization of dorsal horn interneurons. In : Willis WD, editor. Sensory mechanisms of the spinal cord. 3rd ed. Boston: Springer;2004. p. 271–560.

15. Willcockson WS, Chung JM, Hori Y, Lee KH, Willis WD. Effects of iontophoretically released amino acids and amines on primate spinothalamic tract cells. J Neurosci. 1984; 4:732–740.

16. Price TJ, Cervero F, de Koninck Y. Role of cation-chloride-cotransporters (CCC) in pain and hyperalgesia. Curr Top Med Chem. 2005; 5:547–555.

17. Möhler H, Rudolph U, Boison D, Singer P, Feldon J, Yee BK. Regulation of cognition and symptoms of psychosis: focus on GABA(A) receptors and glycine transporter 1. Pharmacol Biochem Behav. 2008; 90:58–64.

18. Schönrock B, Bormann J. Modulation of hippocampal glycine receptor channels by protein kinase C. Neuroreport. 1995; 6:301–304.

19. Vaello ML, Ruiz-Gómez A, Lerma J, Mayor F Jr. Modulation of inhibitory glycine receptors by phosphorylation by protein kinase C and cAMP-dependent protein kinase. J Biol Chem. 1994; 269:2002–2008.

20. Albarran FA, Roa JP, Navarrete R, Castillo R, Nualart F, Aguayo LG. Effect of protein kinase C activation on the glycine evoked Cl(-) current in spinal cord neurons. Brain Res. 2001; 902:1–10.

21. Martin GR, Humphrey PP. Receptors for 5-hydroxytryptamine: current perspectives on classification and nomenclature. Neuropharmacology. 1994; 33:261–273.

22. Xu TL, Nabekura J, Akaike N. Protein kinase C-mediated enhancement of glycine response in rat sacral dorsal commissural neurones by serotonin. J Physiol. 1996; 496(Pt 2):491–501.

23. Li H, Kang JF, Li YQ. Serotonin potentiation of glycine-activated whole-cell currents in the superficial laminae neurons of the rat spinal dorsal horn is mediated by protein kinase C. Brain Res Bull. 2002; 58:593–600.

24. Nguyen HT, Bhattarai JP, Park SJ, Lee JC, Cho DH, Han SK. Enhanced GABA action on the substantia gelatinosa neurons of the medullary dorsal horn in the offspring of streptozotocin-injected mice. J Diabetes Complications. 2015; 29:629–636.

25. Hoyer D, Hannon JP, Martin GR. Molecular, pharmacological and functional diversity of 5-HT receptors. Pharmacol Biochem Behav. 2002; 71:533–554.

26. Boess FG, Martin IL. Molecular biology of 5-HT receptors. Neuropharmacology. 1994; 33:275–317.

27. Yang EJ, Han SK, Park SJ. Functional expression of 5-HT₇ receptor on the substantia gelatinosa neurons of the trigeminal subnucleus caudalis in mice. Brain Res. 2014; 1543:73–82.

28. Yoshimura M, Nishi S. Primary afferent-evoked glycine- and GABA-mediated IPSPs in substantia gelatinosa neurones in the rat spinal cord in vitro. J Physiol. 1995; 482(Pt 1):29–38.

29. Sorkin LS, McAdoo DJ, Willis WD. Raphe magnus stimulation-induced antinociception in the cat is associated with release of amino acids as well as serotonin in the lumbar dorsal horn. Brain Res. 1993; 618:95–108.

30. Langosch D, Becker CM, Betz H. The inhibitory glycine receptor: a ligand-gated chloride channel of the central nervous system. Eur J Biochem. 1990; 194:1–8.

31. Swope SL, Moss SJ, Blackstone CD, Huganir RL. Phosphorylation of ligand-gated ion channels: a possible mode of synaptic plasticity. FASEB J. 1992; 6:2514–2523.

32. Grenningloh G, Pribilla I, Prior P, Multhaup G, Beyreuther K, Taleb O, Betz H. Cloning and expression of the 58 kd beta subunit of the inhibitory glycine receptor. Neuron. 1990; 4:963–970.

33. Béchade C, Sur C, Triller A. The inhibitory neuronal glycine receptor. Bioessays. 1994; 16:735–744.

34. Raymond LA, Blackstone CD, Huganir RL. Phosphorylation of amino acid neurotransmitter receptors in synaptic plasticity. Trends Neurosci. 1993; 16:147–153.

35. Xu TL, Pang ZP, Li JS, Akaike N. 5-HT potentiation of the GABA(A) response in the rat sacral dorsal commissural neurones. Br J Pharmacol. 1998; 124:779–787.

36. Li H, Lang B, Kang JF, Li YQ. Serotonin potentiates the response of neurons of the superficial laminae of the rat spinal dorsal horn to gamma-aminobutyric acid. Brain Res Bull. 2000; 52:559–565.

37. Aghajanian GK, Marek GJ. Serotonin, via 5-HT_2A receptors, increases EPSCs in layer V pyramidal cells of prefrontal cortex by an asynchronous mode of glutamate release. Brain Res. 1999; 825:161–171.

38. Wang D, Li YQ, Li JL, Kaneko T, Nomura S, Mizuno N. γ-aminobutyric acid- and glycine-immunoreactive neurons postsynaptic to substance P-immunoreactive axon terminals in the superficial layers of the rat medullary dorsal horn. Neurosci Lett. 2000; 288:187–190.

39. Edelman AM, Blumenthal DK, Krebs EG. Protein serine/threonine kinases. Annu Rev Biochem. 1987; 56:567–613.

40. Breitinger U, Bahnassawy LM, Janzen D, Roemer V, Becker CM, Villmann C, Breitinger HG. PKA and PKC modulators affect ion channel function and internalization of recombinant alpha1 and alpha1-beta glycine receptors. Front Mol Neurosci. 2018; 11:154.

41. Han L, Talwar S, Wang Q, Shan Q, Lynch JW. Phosphorylation of α3 glycine receptors induces a conformational change in the glycine-binding site. ACS Chem Neurosci. 2013; 4:1361–1370.

42. Gentet LJ, Clements JD. Binding site stoichiometry and the effects of phosphorylation on human alpha1 homomeric glycine receptors. J Physiol. 2002; 544(Pt 1):97–106.

43. Huang R, He S, Chen Z, Dillon GH, Leidenheimer NJ. Mechanisms of homomeric alpha1 glycine receptor endocytosis. Biochemistry. 2007; 46:11484–11493.

44. Song YM, Huang LY. Modulation of glycine receptor chloride channels by cAMP-dependent protein kinase in spinal trigeminal neurons. Nature. 1990; 348:242–245.

45. Gu Y, Huang LY. Cross-modulation of glycine-activated Cl- channels by protein kinase C and cAMP-dependent protein kinase in the rat. J Physiol. 1998; 506(Pt 2):331–339.

46. Agopyan N, Tokutomi N, Akaike N. Protein kinase A-mediated phosphorylation reduces only the fast desensitizing glycine current in acutely dissociated ventromedial hypothalamic neurons. Neuroscience. 1993; 56:605–615.

47. Uchiyama M, Hirai K, Hishinuma F, Akagi H. Down-regulation of glycine receptor channels by protein kinase C in Xenopus oocytes injected with synthetic RNA. Brain Res Mol Brain Res. 1994; 24:295–300.

48. Worley PF, Baraban JM, Snyder SH. Heterogeneous localization of protein kinase C in rat brain: autoradiographic analysis of phorbol ester receptor binding. J Neurosci. 1986; 6:199–207.

49. Nagashima T, Tanabe H, Fujioka Y, Nagashima K, Hidaka H. Immunohistochemical localization of protein kinase C isozymes in human cerebellum, hippocampus and spinal cord. Acta Histochem Cytochem. 1991; 24:441–446.

50. Lynch JW. Molecular structure and function of the glycine receptor chloride channel. Physiol Rev. 2004; 84:1051–1095.

51. Ruiz-Gómez A, Vaello ML, Valdivieso F, Mayor F Jr. Phosphorylation of the 48-kDa subunit of the glycine receptor by protein kinase C. J Biol Chem. 1991; 266:559–566.

52. Malosio ML, Grenningloh G, Kuhse J, Schmieden V, Schmitt B, Prior P, Betz H. Alternative splicing generates two variants of the alpha 1 subunit of the inhibitory glycine receptor. J Biol Chem. 1991; 266:2048–2053.

53. Specht CG, Grünewald N, Pascual O, Rostgaard N, Schwarz G, Triller A. Regulation of glycine receptor diffusion properties and gephyrin interactions by protein kinase C. EMBO J. 2011; 30:3842–3853.

54. Doly S, Madeira A, Fischer J, Brisorgueil MJ, Daval G, Bernard R, Vergé D, Conrath M. The 5-HT_2A receptor is widely distributed in the rat spinal cord and mainly localized at the plasma membrane of postsynaptic neurons. J Comp Neurol. 2004; 472:496–511.

55. Morales M, Battenberg E, Bloom FE. Distribution of neurons expressing immunoreactivity for the 5HT3 receptor subtype in the rat brain and spinal cord. J Comp Neurol. 1998; 402:385–401.

56. Pompeiano M, Palacios JM, Mengod G. Distribution and cellular localization of mRNA coding for 5-HT_1A receptor in the rat brain: correlation with receptor binding. J Neurosci. 1992; 12:440–453.

57. Yin H, Park SA, Han SK, Park SJ. Effects of 5-hydroxytryptamine on substantia gelatinosa neurons of the trigeminal subnucleus caudalis in immature mice. Brain Res. 2011; 1368:91–101.

58. Grudt TJ, Williams JT, Travagli RA. Inhibition by 5-hydroxytryptamine and noradrenaline in substantia gelatinosa of guinea-pig spinal trigeminal nucleus. J Physiol. 1995; 485(Pt 1):113–120.

59. Xie DJ, Uta D, Feng PY, Wakita M, Shin MC, Furue H, Yoshimura M. Identification of 5-HT receptor subtypes enhancing inhibitory transmission in the rat spinal dorsal horn in vitro. Mol Pain. 2012; 8:58.

60. Kobilka BK, Frielle T, Collins S, Yang-Feng T, Kobilka TS, Francke U, Lefkowitz RJ, Caron MG. An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins. Nature. 1987; 329:75–79.

61. Middlemiss DN, Fozard JR. 8-Hydroxy-2-(di-n-propylamino)-tetralin discriminates between subtypes of the 5-HT₁ recognition site. Eur J Pharmacol. 1983; 90:151–153.

62. Raymond JR, Mukhin YV, Gelasco A, Turner J, Collinsworth G, Gettys TW, Grewal JS, Garnovskaya MN. Multiplicity of mechanisms of serotonin receptor signal transduction. Pharmacol Ther. 2001; 92:179–212.

63. Abe K, Kato G, Katafuchi T, Tamae A, Furue H, Yoshimura M. Responses to 5-HT in morphologically identified neurons in the rat substantia gelatinosa in vitro. Neuroscience. 2009; 159:316–324.

64. Ito A, Kumamoto E, Takeda M, Shibata K, Sagai H, Yoshimura M. Mechanisms for ovariectomy-induced hyperalgesia and its relief by calcitonin: participation of 5-HT_1A-like receptor on C-afferent terminals in substantia gelatinosa of the rat spinal cord. J Neurosci. 2000; 20:6302–6308.

65. Kroeze WK, Kristiansen K, Roth BL. Molecular biology of serotonin receptors structure and function at the molecular level. Curr Top Med Chem. 2002; 2:507–528.

66. Pazos A, Cortés R, Palacios JM. Quantitative autoradiographic mapping of serotonin receptors in the rat brain. II. Serotonin-2 receptors. Brain Res. 1985; 346:231–249.

67. Sasaki M, Obata H, Saito S, Goto F. Antinociception with intrathecal alpha-methyl-5-hydroxytryptamine, a 5-hydroxytryptamine 2A/2C receptor agonist, in two rat models of sustained pain. Anesth Analg. 2003; 96:1072–1078.

68. Seyrek M, Kahraman S, Deveci MS, Yesilyurt O, Dogrul A. Systemic cannabinoids produce CB1-mediated antinociception by activation of descending serotonergic pathways that act upon spinal 5-HT₇ and 5-HT_2A receptors. Eur J Pharmacol. 2010; 649:183–194.

69. Conn PJ, Sanders-Bush E, Hoffman BJ, Hartig PR. A unique serotonin receptor in choroid plexus is linked to phosphatidylinositol turnover. Proc Natl Acad Sci U S A. 1986; 83:4086–4088.

70. Cox DA, Cohen ML. 5-HT_2B receptor signaling in the rat stomach fundus: dependence on calcium influx, calcium release and protein kinase C. Behav Brain Res. 1996; 73:289–292.

71. Bradley PB, Engel G, Feniuk W, Fozard JR, Humphrey PP, Middlemiss DN, Mylecharane EJ, Richardson BP, Saxena PR. Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptamine. Neuropharmacology. 1986; 25:563–576.

72. Miquel MC, Emerit MB, Nosjean A, Simon A, Rumajogee P, Brisorgueil MJ, Doucet E, Hamon M, Vergé D. Differential subcellular localization of the 5-HT₃-As receptor subunit in the rat central nervous system. Eur J Neurosci. 2002; 15:449–457.

73. Tecott LH, Maricq AV, Julius D. Nervous system distribution of the serotonin 5-HT₃ receptor mRNA. Proc Natl Acad Sci U S A. 1993; 90:1430–1434.

74. Färber L, Haus U, Späth M, Drechsler S. Physiology and pathophysiology of the 5-HT₃ receptor. Scand J Rheumatol Suppl. 2004; 119:2–8.

75. Ruat M, Traiffort E, Leurs R, Tardivel-Lacombe J, Diaz J, Arrang JM, Schwartz JC. Molecular cloning, characterization, and localization of a high-affinity serotonin receptor (5-HT₇) activating cAMP formation. Proc Natl Acad Sci U S A. 1993; 90:8547–8551.

76. Lovenberg TW, Baron BM, de Lecea L, Miller JD, Prosser RA, Rea MA, Foye PE, Racke M, Slone AL, Siegel BW, Danielson PE, Sutcliffe JG, Erlander MG. A novel adenylyl cyclase-activating serotonin receptor (5-HT₇) implicated in the regulation of mammalian circadian rhythms. Neuron. 1993; 11:449–458.

77. Bard JA, Zgombick J, Adham N, Vaysse P, Branchek TA, Weinshank RL. Cloning of a novel human serotonin receptor (5-HT₇) positively linked to adenylate cyclase. J Biol Chem. 1993; 268:23422–23426.

78. Hedlund PB, Kelly L, Mazur C, Lovenberg T, Sutcliffe JG, Bonaventure P. 8-OH-DPAT acts on both 5-HT_1A and 5-HT₇ receptors to induce hypothermia in rodents. Eur J Pharmacol. 2004; 487:125–132.