急性分离的脊髓腹角神经元膜片钳记录技术

摘要

目的:建立急性分离的脊髓腹角神经元膜片钳记录技术以深入研究脊髓腹角神经元上受体的作用.方法:选用6~11 d的新生SD大鼠,麻醉后分离出含腰骶膨大的脊髓,制备切片,给予酶消化,切除背角,将腹角吹打分离出单细胞,进行膜片钳记录.结果:①分离的腹角神经元形态良好,胞体呈纺锤形、三角形和多边形伴多条细长突起;②7个神经元记录到自发动作电位,其细胞电生理特性为:静息电位(-61.88±16.70)mV;阈电位(-47.39±8.02)mV;锋电位幅值(55.79±17.17)mV;超射(8.39±7.70)mV;放电频率(13.54±9.97)Hz;③7个神经元给予1 mmol/L谷氨酸诱发电流幅值(324.56±90.54)pA,翻转电位(19.43±12.10)mV;8个神经元给予2 mmol/L尼古丁诱发电流幅值(296.91±77.44)pA,翻转电位(29.06±14.88)mV.结论:急性分离脊髓腹角神经元膜片钳记录技术切实可行,可用于脊髓腹角神经元相关调质或药物的作用机理研究.%Objective:To establish a technique of patch-clamp recording of acutely dissociated spinal cord ventral horn neurons for further investigation of roles of diverse receptors on the neurons. Methods:Neonatal SD rats(aged 6 to 11 days) were included and received lumbosacral laminectomy after anes-thesia. Then the lumbosacral spinal cord was removed and transverse slices were prepared. Dorsal horn of the slice was cut off after digestion with Papain. Patch-clamp recording was then performed on mechanically dissociated ventral horn neurons. Results:①Dissociated ventral horn neurons had characteris-tic morphology with a fusiform, triangular, or polygonal soma and broad dendrites emanating from the poles;②Electrophysiological parameters for sponta-neous firings of the dissociated neurons (n=7)were:resting potential, (-61.88±16.70) mV;threshold, (-47.39±8.02) mV;spike amplitude, (55.79 ±17.17) mV; overshoot, (8.39±7.70) mV; and firing frequency, (13.54±9.97)Hz; ③The amplitudes of 1 mmol/L glutamate-induced currents recorded in the 7 ventral horn neurons were (324.56±90.54) pA, the average equilibrium potentials (EGlus), (19.43±12.10) mV, whereas amplitudes of 2 mmol/L nicotine-induced currents in the 8 ventral horn neurons were (296.91±77.44) pA, the average equilibrium potentials (ENics),(29.06± 14.88) mV. Conclusion: The whole-cell patch-clamp recording technique for acutely dissociated spinal cord ventral horn neurons was successfully estab-lished, and this technique can be applied to investigate the receptors and their distributions, classifications and functional mechanisms underlying related modulators or drugs.