Hanna W J, Johnson E C, Chaves D, Renninger G H
Department of Physics, University of Guelph, Ontario, Canada.
Vis Neurosci. 1993 Jul-Aug;10(4):609-20. doi: 10.1017/s0952523800005319.
A combination of enzymatic digestions and mechanical disruption was used to isolate photoreceptor cells from the compound lateral eye of the horseshoe crab, Limulus polyphemus. The cells were maintained in a culture medium and tested for function using whole-cell and cell-attached patch configurations of the gigaseal technique. The cells dissociated from the eye generated spontaneous voltage and current bumps in the dark, and depolarized in a graded fashion to increasing intensities of light over several decades, producing responses similar to those of cells in vivo. Currents evoked during voltage clamp were similar to those in ventral photoreceptor cells of Limulus, although transient currents in the dark- and light-activated currents were smaller in isolated lateral eye cells, perhaps because of the slow speed and spatial nonuniformity of the clamp in these large cells. In addition to isolated cells, dissociation of the compound eye produced small clusters of cells and isolated ommatidia which were also tested for function. Comparison of the electrical characteristics of isolated cells with those of cells in small clusters and in their ommatidial matrix suggests that the electrical junctions normally connecting photoreceptor cells within an ommatidium are functional in the latter groups, but not in isolated cells. Cell-attached patches of rhabdomeral membrane of isolated cells contained light-activated channels, resembling those observed in ventral photoreceptor cells, but no voltage-activated channels. Similar patches of arhabdomeral membrane contained voltage-activated channels, but no light-activated channels. We conclude that this preparation is suitable for studies of processes involved in generating the light response in invertebrate photoreceptor cells.
采用酶消化和机械破碎相结合的方法，从鲎（美洲鲎）的复眼中分离出感光细胞。将这些细胞置于培养基中，并使用千兆封接技术的全细胞和细胞贴附式膜片钳配置来测试其功能。从眼中分离出的细胞在黑暗中产生自发的电压和电流波动，并在几十年内随着光照强度的增加以分级方式去极化，产生与体内细胞相似的反应。电压钳制期间诱发的电流与鲎腹侧感光细胞中的电流相似，尽管在分离的侧眼细胞中，黑暗和光激活电流中的瞬态电流较小，这可能是由于这些大细胞中钳制的速度较慢和空间不均匀性所致。除了分离的细胞外，复眼的解离还产生了小细胞簇和分离的小眼，也对它们进行了功能测试。将分离细胞的电特性与小细胞簇及其小眼基质中的细胞的电特性进行比较表明，通常连接小眼内感光细胞的电连接在后者中起作用，但在分离细胞中不起作用。分离细胞的视杆膜的细胞贴附膜片含有光激活通道，类似于在腹侧感光细胞中观察到的通道，但没有电压激活通道。类似的无视杆膜片含有电压激活通道，但没有光激活通道。我们得出结论，这种制备方法适用于研究无脊椎动物感光细胞中产生光反应所涉及的过程。
