可视膜片钳法记录初级传入纤维介导的脊髓背角神经元突触后电流(英文)  

Visually guided patch-clamp recording of spinal dorsal horn neuron's postsynaptic current evoked by primary afferent fiber

作　　者：万业宏[1] 王玉英[1] 戴飞[1] 胡三觉[1] 

机构地区：[1]第四军医大学基础部神经科学研究所,西安710033

出　　处：《生理学报》2004年第4期550-557,共8页Acta Physiologica Sinica

基　　金：This work was supported by the National Natural Science Foundation of China (No.30030040) ; the National Basic Research Priorities Programme of China (G1999054000).

摘　　要：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。The authors describe here the procedures for using the gelatin half-embedding method to obtain thin spinal cord slices with attached dorsal roots and performing visually guided whole-cell patch-clamp recording of postsynaptic currents evoked by primary afferent fibers in rat spinal dorsal horn. A segment of spinal cord with attached dorsal roots was prepared and half-embedded in an agar block with 20% (w/v) gelatin. Thin spinal cord slices with attached dorsal roots were obtained with a vibratome and whole-cell patch-clamp configuration was established under the infrared observation. At the holding potential of -70 mV, spontaneous excitatory postsynaptic currents (EPSCs) and dorsal root stimulation-evoked EPSCs were recorded as inward currents. According to the conduction velocity of afferent fibers and stimulus threshold, evoked EPSCs that are mediated by A-like or C-like fibers were distinguished. At the holding potential of 0 mV, spontaneous inhibitory postsynaptic currents (IPSCs) and dorsal root stimulation-evoked IPSCs were recorded as outward currents. Using 5 μmol/L strychnine or 20 μmol/L bicuculline, GABAergic or glycinergic evoked IPSCs could be isolated. Using visual patch-clamp method synaptic transmission can be accurately assessed by measuring postsynaptic currents of the dorsal horn neurons. More importantly, with the aid of infrared observation, the incidence of failure to establish a clamp configuration can be greatly reduced and it becomes easier to make recordings from the neurons in deep dorsal horn laminae. Thus, the present research approach an effective approach to study the modulation of primary afferent synaptic transmission.

关 键 词：膜片钳技术 兴奋性突触后电位 抑制性突触后电位 初级传入突触 脊髓 背角 大鼠 

分 类 号：R33[医药卫生—人体生理学]