Orexin-A对脊髓腹角神经元基本电生理参数及尼古丁电流的作用

Effects of orexin-A on basic electrophysiological parameters and nicotinic current in spinal ventral-horn neurons

目的:研究orexin-A对脊髓腹角神经元基本电生理参数及烟碱型乙酰胆碱受体(nAChR)的作用。方法:使用7~12d的新生SD大鼠。麻醉后,将含有腰骶膨大的脊髓分离并切片。用木瓜蛋白酶(papain,0.18g/30mL人工脑脊液消化切片并孵育40min。显微镜下选取腹角,使用抛光的巴斯德吸管进行神经元的急性机械分离。对贴壁的健康神经元进行穿孔膜片钳记录。结果:①急性分离的脊髓腹角神经元状态良好,具有形状多样的胞体和完整的突起;②9个急性分离的脊髓腹角神经元有自发动作电位;③持续灌流orexin-A(OXA)2min后,在5例细胞记录到4个脊髓腹角神经元的自发动作电位放电频率增加了(63.64±5.25)%,幅度等参数无明显变化;④在15个神经元,有12例细胞给予0.3mmol/L尼古丁诱导出内向电流,幅度为(142.97±75.02)pA,用100nmol/LOXA进行2min的预处理,可显著抑制电流幅度至(69.07±61.07)pA(P<0.001),抑制率为(54.75±22.62)%;⑤在9个神经元中有7例细胞给予尼古丁诱导的电流幅度为(129.31±69.38)pA,将100nmol/LOXA共同施用于神经元,尼古丁诱导的电流幅度降为(68.61±34.98)pA,在此基础上,通过施用orexin-1受体(OX1R)拮抗剂SB334867(10μmol/L)2min后可阻断OXA对尼古丁诱导电流的抑制作用,电流幅值为(93.46±56.45)pA。因此,SB334867可完全取消OXA对尼古丁电流的抑制作用。但对于另两例神经元,SB334867并未阻断OXA的抑制作用。结论:Orexin-A可能通过OX1R对脊髓大部分腹角神经元的尼古丁电流具有抑制作用,但不能排除orexin-2受体(OX2R)也参与OXA作用的可能性。

Objective:To investigate the effects of orexin-A on basic electrophysiological parameters and nicotinic acetylcholine receptors (nAChRs) in spinal ventral-horn neurons.Methods:Neonatal SD rats,aged 7-12 days,were used as experimental animals.After anesthesia,the spinal cord containing the lumbosacral enlargement was separated and sliced.The slices were digested with digestive enzyme (Papain,0.18 g/30 mL artificial cerebrospinal fluid,ACSF) and incubated for 40 minutes.The ventral horn was selected for acute mechanical dissociation of neurons with fire-polished micro-Pasteur pipette.Single cells were dissociated and perforated patch-clamp recording was performed.Results:①The isolated ventral horn neurons were in good condition with large diverse somata and intact processes;②The spontaneous action potentials of 9 acutely isolated spinal ventral horn neurons were recorded;③After 2 minutes of continuous perfusion of orexin-A (OXA),the spontaneous action potential discharge frequency of the four spinal ventral horn neurons was increased (63.64±5.25)%,with no significant change in other parameters,such as amplitude;④In 12 of the 15 neurons,0.3 mmol/L nicotine induced inward current with amplitude of (142.97±75.02) pA,and bath application with 100 nmol/L orexin-A for 2 minutes significantly inhibited the current amplitude to (69.07±61.07) pA ( P< 0.001),that is,inhibition ratio was (54.75±22.62)%,and ⑤ In 7 of the 9 neurons,the nicotine-induced current amplitude was (129.31±69.38) pA,100 nmol/L orexin-A was co-administered to the neurons,and the nicotine-induced current amplitude decreased to (68.61±34.98) pA.On this basis,the inhibitory effect of OXA on nicotine-induced current was blocked by administration of the orexin-1 receptor (OX1R) antagonist SB334867 (10 μmol/L) for 2 minutes.The current amplitude was (93.46±56.45) pA.Therefore,SB334867 completely nullified the inhibition of nicotine current by orexin A.But for the other two neurons,SB334867 did not prevent the inhibition of orexin A.Conclusion:Orexin-A,probably via OX1Rs,has an inhibitory action on nicotine currents in most of ventral horn neurons in spinal cord,but the possibility of OX2R involvement in the orexin-A effects cannot be excluded either.