摘要
目的:探究食欲素-A(orexin-A)和α_(4)β_(2)-N型乙酰胆碱受体(α_(4)β_(2)-nAChR)选择性激动剂RJR-2403分别对新生大鼠离体脊髓腹角神经元自发动作电位影响的差异。方法:应用8~12 d新生SD大鼠,麻醉后游离出颈端至骶部的脊髓,保留腰骶膨大节段并制备切片。切片孵育30min,采用木瓜蛋白酶消化19~22 min,转常温孵育45~60 min。沿中央管切取脊髓腹角,使用口径从大到小的巴斯德吸管急性机械分离神经元,静置20 min待细胞贴壁,在倒置显微镜下利用膜片钳记录良好的神经元,联合药理学方法进行功能性研究。在电流钳记录模式下,观察orexin-A和RJR-2403分别对离体脊髓腹角神经元自发放电(动作电位)的影响,并比较多种参数的差异。结果:(1)本研究应用急性分离的脊髓腹角神经元进行膜片钳记录,所记录的神经元胞体完整,形态多样,能发出较多突起且有分支;(2)给予orexin-A前的腹角神经元自发动作电位的频率[(3.55±0.66)Hz]低于给药后[(8.26±3.31)Hz](P<0.01),给药前动作电位幅值[(95.28±7.21)mV]高于给药后[(85.15±5.80)mV](P<0.05),静息电位和动作电位半幅时程无明显变化;(3)应用RJR-2403前,腹角神经元的静息电位[(-66.67±43.38)mV]高于给药后[(-60.48±3.38)mV](P<0.01),给药前放电频率[(2.74±1.60)Hz]低于给药后[(8.94±3.14)Hz](P<0.001),给药前动作电位幅值[(84.96±4.85)mV]高于给药后[(73.77±5.42)mV](P<0.01),给药前半幅时程[(4.34±1.52)ms]低于给药后[(5.39±1.80)ms](P<0.01)。结论:Orexin-A和RJR-2403对脊髓腹角神经元自发动作电位均具有正性调制作用,但对于静息电位、半幅时程存在不同作用,可能源于促代谢型受体和促离子型受体介导的作用差异。
Objective:To investigate different effects of orexin-A and α_(4)β_(2)-N-type acetylcholine receptor(α_(4)β_(2)-nAChR)selective agonist RJR-2403 on the spontaneous action potentials of isolated spinal ventral horn neurons in neonatal rats.Methods:The spinal cord from cervical to sacral segments was initially dissociated from neonatal Sprague-Dawley rats(8-12 days old)after anesthesia,and the lumbosacral enlargement segment was preserved to prepare slices.The slices were incubated for 30 min,digested with papain for 19-22 min and incubated at room temperature for 45-60 min.The ventral horn of the spinal cord was excised along the central canal,and the neurons were acutely mechanically dissociated using Pasteur pipettes of large to small calibers and left for 20 min for cell adhesion.Good neurons were recorded using patch clamp under inverted microscope,and functional studies were conducted by patch-clamp recordings combined with pharmacological methods.Under the current-clamp recording mode,the effects of orexin-A and RJR-2403 on the spontaneous action potential of spinal ventral horn neurons in vitro were observed,and the differences of various parameters were compared.Results:①In this experiment,the acutely isolated spinal ventral horn neurons were recorded by patch-clamp recordings.The recorded neurons had intact somata,diverse morphologically,and could emit many protrusions with branches;②The spontaneous action potential frequency of ventral horn neurons was increased significantly from the initial(3.55±0.66)Hz to(8.26±3.31)Hz after application of orexin-A(P<0.01),and action potential amplitude was decreased from the initial(95.28±7.21)mV to(85.15±5.80)mV(P<0.05),yet no significant changes were observed in the half-amplitude duration of resting potential and action potential;③After application of RJR-2403,the resting potential of ventral horn neurons depolarized from the initial(-66.67±43.38)mV to(-60.48±3.38)mV(P<0.01).The firing frequency was significantly accelerated from the initial(2.74±1.60)Hz to(8.94±3.14)Hz(P<0.001),whereas the action potential amplitude was notably reduced from the initial(84.96±4.85)mV to(73.77±5.42)mV(P<0.01),and the half-width was increased from(4.34±1.52)ms to(5.39±1.80)ms(P<0.01).Conclusion:Both orexin-A and RJR-2403 have positive modulatory effects on spontaneous action potentials in spinal ventral horn neurons,yet produce different effects on the resting potentials and half-width,which is probably due to the differences in the effects mediated by metabotropic receptors and ionotropic receptors.
作者
储婉玉
李妍
甄骋
盛鑫
何光侣
徐爱萍
张环环
郑超
CHU Wanyu;LI Yan;ZHEN Cheng;SHENG Xin;HE Guanglü;XU Aiping;ZHANG Huanhuan;ZHENG Chao(Department of Neurobiology,Wannan Medical College,Wuhu 241002,Anhui,China)
出处
《皖南医学院学报》
CAS
2024年第2期103-107,共5页
Journal of Wannan Medical College
基金
国家自然科学基金项目(31200828)
安徽省自然科学基金面上项目(2108085MC91)
安徽省高校学科拔尖人才学术资助项目(gxbjZD2021061)
安徽省高等学校自然科学研究重点项目(KJ2019A0411)。
