培养大鼠视网膜神经节细胞的形态学与电生理学特性

目的比较不同培养天数大鼠视网膜神经节细胞(retinal ganglion cells,RGCs)形态学特性与电生理功能,探讨其形态与功能的最佳培养时期。方法采用出生后0dLong Evans大鼠,取视网膜培养神经节细胞,分别于培养后第1d、4d、7d3个时间点观察细胞形态,并行膜片钳全细胞记录研究其静息膜电位(resting membrane potential,RMP)和动作电位(action potential,AP)的阈值、幅度及波宽。结果共记录到53个RGCs,根据AP类型RGCs分为单峰型(39个)、瞬变型(11个)和持续型(3个),其中以单峰型为主(占73.6%)。培养1d RGCs不易诱发AP,多数细胞需增加刺激强度后方可诱发出AP;培养4d时绝大多数RGCs给予20PA的刺激即可诱发出AP;培养7d时虽较容易诱发AP,但由于此时细胞存活状态较差,部分细胞(共记录28个细胞,其中8个不能诱发AP)给予不同强度的刺激,均不能诱发出AP。不同培养时期的RGCs静息膜电位和AP的特性无显著差异。结论随着培养时间的延长RGCs形态逐渐趋于成熟,但电生理功能仍处于幼稚状态;培养4d的RGCs形态和电生理状态最好。

Electrophysiological and morphological properties of cultured rat retinal ganglion cells

Objective To compare the electrophysiological and morphological properties of rat retinal ganglion cells (RGCs) during different culture days and to find the optimal culture phase for morphology and function.Methods RGCs during different culture days were examined to observe the changes of morphological properties.Patch-clamp whole cell recordings were adopted.Resting membrane potential (RMP) and threshold,amplitude,wave-width of action potential (AP) evoked by depolarizing currents were recorded for electrophysiological studies in rat RGCs.Results Fifty cultured RGCs were acquired.Three different discharge patterns,single spike (39),transient spike (11) and sustained spike (3) of RGCs in response to maintained depolarizing currents were recorded.Singe spike was the main firing pattern of cultured RGCs.AP was difficult to be evoked in RGCs cultured for 1 day;whereas easy in RGCs cultured for 4 days;easy to be evoked but the electrophysiolgical properties were not very healthy in RGCs cultured for 7 days.There were no significant differences in membrane properties of RGCs during different culture periods.Conclusion The RGCs isolated from Long Evans rats can be successfully cultured and these cultured RGCs keep the similar morphology as those of in vivo.The membrane properties of RGCs are still immature even though these cells become gradually mature during culture days.The electrophysiological and morphological properties of RGCs cultured for 4 days are the best according to the patch-clamp whole cell recordings in our study.