Wang Jin-Shan, Kefalov Vladimir J
Department of Ophthalmology and Visual Sciences, Washington University in St. Louis, School of Medicine, USA.
J Vis Exp. 2010 Jan 5(35):1681. doi: 10.3791/1681.
Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.
视网膜中的视杆和视锥光感受器负责光检测。在黑暗中，外段的环核苷酸门控（CNG）通道开放，允许阳离子稳定地向内流过膜，使细胞去极化。光照会触发CNG通道关闭，阻止内向阳离子电流流动，从而导致细胞超极化。基于光感受器的极性，20世纪70年代开发了一种吸力记录方法，与经典的膜片钳技术不同，该方法不需要穿透质膜。将外段吸入充满细胞外溶液的紧密贴合的玻璃微管中，可以记录单个细胞在测试闪光照射时的电流变化。然而，这种成熟的“外段吸入（OS-in）”吸力记录不适用于小鼠视锥细胞记录，因为小鼠视网膜中视锥细胞的比例较低（3%），且难以识别视锥细胞的外段。最近，开发了一种内段吸入（IS-in）记录配置，将光感受器的内段/核区域吸入记录微管中。在本视频中，我们将展示如何使用单细胞吸力电极记录单个小鼠视锥细胞的光反应。
