Kolesnikov Alexander V, Kefalov Vladimir J
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, Washington, USA.
J Vis Exp. 2012 Mar 14(61):3424. doi: 10.3791/3424.
There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin α-subunit knockout (Tα(-/-)) mice which lack rod signaling(8).
视杆细胞和视锥细胞。视杆细胞能够检测单个光量子，而视锥细胞则在快速变化的强光条件下持续工作。光感受器外段中视杆细胞和视锥细胞特异性视觉色素对光的吸收触发了光转导级联反应，最终导致质膜上的环核苷酸门控通道关闭和细胞超极化。这种光诱导的膜电流和电位变化可以通过经典的吸电极记录技术或通过对分离的视网膜进行药理学阻断突触后反应成分后的经视网膜视网膜电图记录（ERG）来记录为光反应。后一种方法允许从小鼠光感受器进行药物可及的长期记录，并且对于从稀少且脆弱的小鼠视锥细胞获得稳定的光反应特别有用。对于视锥细胞，这样的实验可以在暗适应条件下以及在基本上使所有视觉色素漂白的强光照射后进行，以监测暗适应过程中视锥细胞光敏性恢复的过程。在本视频中，我们将展示如何从暗适应的小鼠视网膜进行视杆细胞和M/L视锥细胞驱动的经视网膜记录。视杆细胞记录将使用野生型（C57Bl/6）小鼠的视网膜进行。为了简单起见，视锥细胞记录将从缺乏视杆细胞信号传导的基因修饰视杆转导蛋白α亚基敲除（Tα(-/-)）小鼠获得(8)。
