Nasi E, Gomez M
Department of Physiology, Boston University School of Medicine, Woods Hole, MA 02118.
Vis Neurosci. 1992 Apr;8(4):349-58. doi: 10.1017/s0952523800005083.
A protocol was developed to isolate enzymatically photoreceptors from the retina of the squid, Loligo pealei. The procedure routinely results in a high yield of intact cells. Examination of solitary photoreceptors under Nomarski optics revealed that the fine morphological features described in anatomical studies of retinal sections are retained. The distal segment is up to 250 microns long, 4-7 microns wide, covered in part by short microvilli; the inner segment and the cell body, with the initial portion of the axon, are also clearly discernible in solitary cells. Suction electrode measurements performed from the cell body confirmed that responsiveness to light survived cell isolation. Macroscopic membrane currents were measured using the whole-cell tight-seal technique, and the perforated-patch method. Step depolarizations of membrane voltage administered in the dark elicited a slowly activating, sustained outward current. Light stimulation evoked an inward current graded with stimulus intensity; the peak current could amply exceed 1000 pA. Intense photostimulation gave rise to a prolonged inward aftercurrent that lasted for tens of seconds. On-cell patch recording along the intermediate segment and most of the smooth areas of the distal segment showed a large incidence of silent patches, with the occasional presence of voltage-dependent channels. On the other hand, channel activity could be recorded more frequently from electrode placements near the apical tip of the cell, where the presence of microvilli could be confirmed visually. Some patches were unresponsive to voltage stimulation applied in the dark but produced distinct bursts of channel openings after illumination. The feasibility of single-cell electrophysiology in isolated photoreceptors, together with the growing body of biochemical information on cephalopod preparations, makes squid an attractive model system to investigate the visual process in invertebrates using multiple experimental approaches.
我们制定了一项方案，用于从枪乌贼（Loligo pealei）的视网膜中酶解分离光感受器。该程序通常能产生高产量的完整细胞。在Nomarski光学显微镜下观察单个光感受器发现，视网膜切片解剖学研究中描述的精细形态特征得以保留。远端部分长达250微米，宽4 - 7微米，部分覆盖着短微绒毛；在单个细胞中，内段、细胞体以及轴突的起始部分也清晰可辨。从细胞体进行的吸电极测量证实，细胞分离后对光的反应性依然存在。使用全细胞紧密封技术和穿孔膜片法测量宏观膜电流。在黑暗中施加的膜电压阶跃去极化引发缓慢激活的持续外向电流。光刺激引发与刺激强度成比例的内向电流；峰值电流可充分超过1000 pA。强烈的光刺激会产生持续数十秒的内向后电流。沿中间段和远端段大部分平滑区域进行的膜片钳记录显示，沉默膜片的发生率很高，偶尔会出现电压依赖性通道。另一方面，在细胞顶端附近的电极位置更频繁地记录到通道活性，在这些位置通过视觉可确认微绒毛的存在。一些膜片在黑暗中施加电压刺激时无反应，但在光照后会产生明显的通道开放爆发。分离的光感受器中单细胞电生理学的可行性，以及关于头足类动物制剂的生化信息不断增加，使得枪乌贼成为一个有吸引力的模型系统，可使用多种实验方法研究无脊椎动物的视觉过程。
