摘要
目的:探讨低温琼脂包埋振动切片机切片法制作的大鼠视网膜切片内核层神经元的形态和基本电生理学特性。方法:采用低温琼脂包埋振动切片机切片的方法制作大鼠视网膜切片,对内核层的神经元进行膜片钳全细胞记录,同时在胞内液中加入荧光黄观察记录细胞的形态。结果:该方法制作的视网膜切片切面平整、细胞活性好、保留了细胞之间的突起联系,能够根据细胞胞体的大小、位置初步辨别细胞的种类。在视网膜切片上荧光黄显示的细胞形态表明,双极细胞胞体呈梭形,突起主要沿纵向延伸;而水平细胞和无长突细胞胞体圆形或椭圆形、胞体较大,分别位于内核层的最外层和最内层。水平细胞和无长突细胞的静息膜电位(RMP)和膜电容(Cm)明显高于双极细胞。给予时程40ms,步阶10mV从-60mV至+40mV的电压刺激,41.7%的视锥双极细胞和64.7%的无长突细胞表现出内向钠电流和外向钾电流,其他细胞则只表现出外向钾电流。结论:采用低温琼脂包埋振动切片机切片的方法操作简单,制作的切片质量稳定可靠,使得在视网膜切片上对包括水平细胞在内的不同内核层神经元进行膜片钳记录成为可能。进一步研究视网膜内核层神经元的电生理学特性,有助于揭示视觉信号的发生、传导和调控机制。
·AIM:To investigate the morphology and electrophysiological properties of neurons in the inner nuclear layer(INL)of the adult rat retina slices.The retinal slices were prepared by low-melt agarose embedding and then cutted by vibratome.·METHODS:Whole cell patch clamp and intracellular staining with Lucifer Yellow were used in this study to study the morphology and electrophysiological properties of the INL neurons in retinal slices.·RESULTS:Retinal slices prepared in this method possessed a very smooth surface.The cells on the retinal slices maitained very good vitality,and some of the cells even retained their dendritic connections with other cells on the slice.According to the size and location of the cell bodies,neurons in the INL were easy to differentiate.Luciifer Yellow contained in the intracellular solution revealed the morphology of the recorded cell very well.Bipolar cells possessed elongated cell bodies and their processes mainly extended along the vertical direction.Horizontal cells and amacrine cells possessed much bigger and round cell bodies,resided in the outermost and inner most of the INL,respectively.The rest membrane potential and membrane capacitance of horizontal cell and amacrine cell were much higher than that of bipolar cells.Under a voltage step from-60mV to+40mV,10mV per step,41.7%of the cone bipolar cells and 64.7%of the amacrine cells exhibited inward sodium current and outward potassium current.Other cells only possessed outward potassium current.·CONCLUSION:The method of preparing retinal slices was very simple,and the viability of the slices were stable.These facilitated the patch-clamp recording of all the neurons in the INL including horizontal cells.Further investigation of the electrophysiological properties of the neurons in the INL was essential in revealing the mechanism of vision.
作者
黄艳明
杨梅
袁容娣
Yan-Ming Huang;Mei Yang;Rong-Di Yuan(Department of Ophthalmology, the Second Affiliated Hospital of Army Medical University, Chongqing 400037, China)
出处
《国际眼科杂志》
CAS
北大核心
2020年第5期778-781,共4页
International Eye Science
基金
国家自然科学基金资助项目(No.81600758)。
关键词
视网膜切片膜片钳
内核层
细胞内染色
全细胞记录
retina slice patch clamp
inner nuclear layer
intracellular staining
whole cell recording
