Orexin-A通过激活OX_(1)R、OX_(2)R和非Ca^(2+)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  

Orexin-A inhibitsγ-aminobutyric acid current of neonatal rat spinal cord ventral horn neurons by activating OX_(1)R,OX_(2)R and Ca^(2+)-independent PKC

作　　者：杨鑫宇 朱苏月 靳娜 李妍 甄骋 张环环[2] 徐爱萍 汪萌芽[3] 郑超[1] YANG Xinyu;ZHU Suyue;JIN Na;LI Yan;ZHEN Cheng;ZHANG Huanhuan;XU Aiping;WANG Mengya;ZHENG Chao(Neurobiology Laboratory,Institute of Physiological Sciences,Wannan Medical College,Wuhu 241002,China;Psychophysiology Laboratory,Institute of Physiological Sciences,Wannan Medical College,Wuhu 241002,China;Cell Electrophysiology Laboratory,Institute of Physiological Sciences,Wannan Medical College,Wuhu 241002,China)

机构地区：[1]皖南医学院生理科学研究所神经生物学研究室,安徽芜湖241002 [2]皖南医学院生理科学研究所心理生理学研究室,安徽芜湖241002 [3]皖南医学院生理科学研究所细胞电生理研究室,安徽芜湖241002

出　　处：《南方医科大学学报》2021年第5期694-701,共8页Journal of Southern Medical University

基　　金：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)。

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GAB A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GAB A电流的压抑作用;给予Bis-Ⅳ后,orexin-A不再压抑GAB A电流(P=0.31,n=5);PMA可模拟orexin-A的作用,显著抑制GAB A电流,抑制率为(60.79±10.94)%,在此基础上,orexin-A不能进一步压抑GAB A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。Objective To investigate the effect of orexin-A on the functionality of ionotropicγ-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 OX_(1)R antagonist),TCSOX_(2)29(a selective OX_(2)R antagonist),Bis-Ⅳ(a PKC inhibitor),PMA(a PKC agonist),Rp-cAMP(a PKA inhibitor),or BAPTA(Ca^(2+)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 TCSOX_(2)29,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 Ca^(2+)-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 ventra

关 键 词：OREXIN-A Orexin受体 脊髓腹角神经元 Γ-氨基丁酸 膜片钳记录 蛋白激酶C 

分 类 号：R338[医药卫生—人体生理学]