南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (5): 694-701.doi: 10.12122/j.issn.1673-4254.2021.05.09

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Orexin-A通过激活OX1R、OX2R和非Ca2+依赖的PKC抑制新生大鼠脊髓腹角神经元的γ-氨基丁酸电流

杨鑫宇，朱苏月，靳 娜，李 妍，甄 骋，张环环，徐爱萍，汪萌芽，郑 超   

  • 出版日期:2021-05-20 发布日期:2021-06-11

Orexin-A inhibits γ-aminobutyric acid current of neonatal rat spinal cord ventral horn neurons by activating OX1R, OX2R and Ca2+-independent PKC

  • Online:2021-05-20 Published:2021-06-11

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摘要： 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。

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

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