[Orexin-A inhibits γ-aminobutyric acid current of neonatal rat spinal cord ventral horn neurons by activating OX 1 R, OX 2 R and Ca 2+-independent PKC]

Nan Fang Yi Ke Da Xue Xue Bao. 2021 May 20;41(5):694-701. doi: 10.12122/j.issn.1673-4254.2021.05.09.
[Article in Chinese]

Abstract

Objective: To investigate the effect of orexin-A on the functionality of ionotropic γ-aminobutyric acid (GABA) receptors in spinal cord ventral horn neurons and its mechanisms.

Objective: The spinal cord containing the lumbosacral enlargement was isolated from neonatal SD rats (7-12 days old) and sliced. The slices were digested with papain (in 0.18 g/30 mL artificial cerebrospinal fluid) for 40-60 min, and the ventral horn neurons were separated acutely using fire-polished Pasteur pipettes. After the cells adhered to the bottom of Petri dishes, patch-clamp experiments combined with pharmacological methods were performed to test the effects of orexin-A on GABA currents of the neurons treated with SB334867 (a selective OX1R antagonist), TCSOX229 (a selective OX2R antagonist), Bis-Ⅳ (a PKC inhibitor), PMA (a PKC agonist), Rp-cAMP (a PKA inhibitor), or BAPTA (Ca2+ chelator).

Objective: The isolated neurons maintained good morphologies with diverse shapes of cell body and long protrusions. Treatment with orexin-A significantly inhibited the amplitude of GABA-induced current (P < 0.001, n=49) with an inhibition rate of (67.48±12.50)%. SB334867 and TCSOX229, when applied simultaneously, completely abolished the suppressive effect of orexin-A on the GABA currents (P=0.93, n=6), and their separate use partially relieved the suppressive effect of orexin-A (P=0.001, n=8; P=0.02, n=8). The application of Bis-Ⅳ also abolished the suppressive effect of orexin-A on GABA currents (P=0.31, n=5). PMA mimicked the effect of orexin-A in these neurons and significantly inhibited GABA currents with an inhibition rate of (60.79±10.94)%, and the application of orexin-A did not cause further suppression of GABA currents in PMA-treated neurons (P=0.15, n=6). Orexin-A was still capable of suppressing GABA currents in Rp-cAMP-treated neurons (P=0.001, n=5). The extracellular Ca2+-free solution (P=0.004, n=8) or the presence of BAPTA (P=0.04, n=7) did not significantly affect the inhibitory effect of orexin-A on GABA currents.

Objective: Orexin-A inhibits GABA currents in the ventral horn neurons of rat spinal cord probably by activating OX1R, OX2R and Ca2+-independent PKC.

目的: 研究orexin-A对脊髓腹角神经元促离子型γ-氨基丁酸(GABA)受体功能的影响及其机制。

方法: 选取7~12 d的新生SD大鼠,麻醉后分离出含腰骶膨大的脊髓节段并切片。使用木瓜蛋白酶(Papain,0.18 g/30 mL人工脑脊液)消化切片并孵育40~60 min。显微镜下保留腹角,使用抛光的不同口径的巴斯德吸管急性机械分离单个细胞,待细胞贴壁后,应用膜片钳记录联合药理学方法对状态良好的神经元开展研究。在电压钳模式下记录,结合预处理给药方式,应用GABA受体激动剂γ-氨基丁酸记录在脊髓腹角神经元诱发的电流,给予orexin-A观察其对GABA电流的调制作用,先后给予OX1R选择性拮抗剂SB334867、OX2R选择性拮抗剂TCSOX229、PKC抑制剂Bis-Ⅳ、PKC激动剂PMA、PKA抑制剂Rp-cAMP、Ca2+螯合剂BAPTA等药物分析orexin-A对GABA电流调制的作用机制。

结果: 分离的脊髓腹角神经元形态良好,胞体形状多样,表面光洁,立体感较强且突起较长;给予orexin-A后,GABA诱发的电流幅值被显著制抑(P < 0.001,n=49),抑制率为(67.48±12.50)%;同时给予SB334867和TCSOX229可完全取消orexin-A对GABA电流的抑制作用(P=0.93,n=6);单独应用SB334867(P=0.001,n=8)或TCSOX229(P=0.02,n=8)均部分解除orexin-A对GABA电流的压抑作用;给予Bis-Ⅳ后,orexin-A不再压抑GABA电流(P=0.31,n=5);PMA可模拟orexin-A的作用,显著抑制GABA电流,抑制率为(60.79±10.94)%,在此基础上,orexin-A不能进一步压抑GABA电流(P=0.15,n=6)。给予Rp-cAMP后,orexin-A仍可显著压抑GABA电流(P=0.001,n=5)。在无Ca2+细胞外液(P=0.004,n=8)或胞内应用BAPTA(P=0.04,n=7)时,orexin-A对GABA电流的压抑作用不受影响。

结论: Orexin-A抑制脊髓腹角神经元的GABA电流,该效应可能经由OX1R和OX2R共同介导以及非Ca2+依赖的PKC信号通路的参与。

Keywords: Orexin-A; orexin receptors; patch-clamp recordings; protein kinase C; spinal ventral horn neurons; γ-aminobutyric acid.

MeSH terms

  • Animals
  • Animals, Newborn
  • Anterior Horn Cells*
  • Neurons
  • Orexins / pharmacology
  • Patch-Clamp Techniques
  • Protein Kinase C
  • Rats
  • Rats, Sprague-Dawley
  • Spinal Cord
  • gamma-Aminobutyric Acid* / pharmacology

Substances

  • Orexins
  • gamma-Aminobutyric Acid
  • calcium-independent protein kinase C
  • Protein Kinase C

Grant support

国家自然科学基金(31200828,31271155);安徽省高校自然科学研究项目(KJ2019A0411,KJ2018A0266);安徽省高校优秀青年人才支持计划项目(gxyqZD2016175,gxyq2017034);安徽省自然科学基金(1908085QC132);皖南医学院博士科研启动基金(rcqd201609)