CAJ | 학술논문

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-70 mV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0mV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5μmol/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.
본문묘술료용명효반포매법제비대배근척수박편적실험보취,화재척수배각기록유초급전입섬유개도적돌촉후전류적가시막편겸법.수술제비일단대배근적척수표본,병용20%적명효포매재경지괴상,재용진동절편궤절편획득대배근적척수박편.통과홍외선가시적인도,재척수배각신경원상건립전세포봉접모식.재겸제전압위-70 mV조건하,기록자발적화배근자격인기적흥강성돌촉후전류.이전입섬유적전도속도여자격역치위지표,가이구분A양섬유여C양섬유흥강성돌촉후전류.재겸제전압위0mV조건하,기록자발적화배근자격인기적억제성돌촉후전류.용5μmol/L적사저혹20μmol/L적하포모단감분리출γ-안기정산능혹감안산능적억제성돌촉후전류.용가시막편겸방법가이준학측량척수배각신경원적돌촉후전류,종이연구초급전입돌촉적전체과정.경중요적시,재홍외선가시관찰적방조하,건립막편겸봉접적성공솔현저제고,동시야사기록연구척수배각심층신경원변득경가용역.본연구위탐색초급전입돌촉전체과정제공료일개유효적방법.
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.