论文部分内容阅读
本文描述了用明胶半包埋法制备带背根脊髓薄片的实验步骤,和在脊髓背角记录由初级传入纤维介导的突触后电流的可视膜片钳法。手术制备一段带背根的脊髓标本,并用20％的明胶包埋在琼脂块上,再用振动切片机切片获得带背根的脊髓薄片。通过红外线可视的引导,在脊髓背角神经元上建立全细胞封接模式。在钳制电压为-70mV条件下,记录自发的和背根刺激引起的兴奋性突触后电流。以传入纤维的传导速度与刺激阈值为指标,可以区分A样纤维与C样纤维兴奋性突触后电流。在钳制电压为0 mV条件下,记录自发的和背根刺激引起的抑制性突触后电流。用5 μmol/L的士宁或20μmol/L的荷包牡丹碱分离出γ-氨基丁酸能或甘氨酸能的抑制性突触后电流。用可视膜片钳方法可以准确测量脊髓背角神经元的突触后电流,从而研究初级传入突触的传递过程。更重要的是,在红外线可视观察的帮助下,建立膜片钳封接的成功率显著提高,同时也使记录研究脊髓背角深层神经元变得更加容易。本研究为探索初级传入突触传递过程提供了一个有效的方法。
This article describes the experimental procedures for the preparation of dorsal spinal cord slices with gelatin semi-embedding and the visual patch clamp method of recording post-synaptic currents mediated by primary afferent fibers in the dorsal horn of the spinal cord. Surgical preparation of a dorsal root with spinal cord specimens, and 20% gelatin embedded in the agar block, and then vibrating microtome slices to obtain spinal cord with dorsal root. Through infrared visual guidance, establishing a whole-cell sealing pattern on the dorsal horn neurons. Excitatory postsynaptic currents induced by spontaneous and dorsal root stimulation were recorded at a clamping voltage of -70 mV. The afferent fiber conduction velocity and stimulation threshold as an index, can distinguish between A-like fibers and C-like fibers excitatory postsynaptic currents. Inhibitory postsynaptic currents induced by spontaneous and dorsal root stimulation were recorded at clamping voltage of 0 mV. Inhibitory postsynaptic currents of gamma-aminobutyric acid or glycine can be isolated with 5 [mu] mol / L strychnine or 20 [mu] mol / L bicuculline. Visual patch clamp method can accurately measure the post-synaptic currents of dorsal horn neurons in order to study the primary afferent synaptic transmission. More importantly, with the help of infrared visual observation, the success rate of establishing patch-clamp closure was significantly improved, and it also made it easier to document deep neurons in the dorsal horn of the spinal cord. This study provides an effective way to explore the primary afferent synaptic transmission.