Jinks R N, Hanna W J, Renninger G H, Chamberlain S C
Institute for Sensory Research, Syracuse University, NY 13244-5290.
Vis Neurosci. 1993 Jul-Aug;10(4):597-607. doi: 10.1017/s0952523800005307.
Isolated photoreceptors are desirable for whole-cell and patch-clamp studies of functional properties of visual processes that cannot be clearly analyzed when the photoreceptors are coupled. The retina of the compound lateral eye of the horseshoe crab, Limulus polyphemus, was dissociated into individual retinular cells using an enzyme pretreatment consisting of collagenase, papain, and trypsin, and a two-stage mechanical dissociation. These photoreceptors are functionally viable in an organ culture medium for up to 1 week and possess naked arhabdomeral and rhabdomeral segment membranes which are easily accessible for whole-cell recordings. A dissection technique was also developed whereby the retinal epidermis and neural plexus, as well as the second-order eccentric cells, could be separated from the ommatidia of the compound lateral eye in one simple step, providing viable isolated ommatidia attached to the cornea. The enzyme pretreatment used for dissociating the retina was then used to remove the individual ommatidia from the corneal cones. Hoffman modulation contrast microscopy was used to develop a reliable method for sorting and collecting viable isolated retinular cells for morphological and electrophysiological studies. Morphological analysis using light microscopy and scanning and transmission electron microscopy revealed that isolated retinular cells are morphologically nearly identical to retinular cells in situ. Isolated retinular cells possess a normal rhabdomere with no apparent loss of microvillar membrane as a result of the isolation process. Ommatidia can presently be isolated with up to six retinular cells possessing essentially normal structure and ultrastructure including thick rays of rhabdom. Isolated ommatidia possess naked A-segment membranes which are also well suited for whole-cell recording techniques.
对于视觉过程功能特性的全细胞和膜片钳研究而言，分离的光感受器是理想的研究对象。当光感受器相互耦联时，这些视觉过程的功能特性难以得到清晰分析。使用由胶原酶、木瓜蛋白酶和胰蛋白酶组成的酶预处理以及两阶段机械解离方法，将美洲鲎复眼侧眼中的视网膜解离为单个小眼细胞。这些光感受器在器官培养基中可保持功能活性长达1周，并具有裸露的无轴突和轴突节段膜，便于进行全细胞记录。还开发了一种解剖技术，通过该技术可以在一个简单步骤中将视网膜表皮和神经丛以及二级偏心细胞从复眼侧眼的小眼分离出来，得到附着于角膜的存活的分离小眼。然后，将用于解离视网膜的酶预处理方法用于从小眼角膜锥中分离出单个小眼。利用霍夫曼调制对比显微镜开发了一种可靠的方法，用于分选和收集存活的分离小眼细胞，以进行形态学和电生理学研究。使用光学显微镜、扫描电子显微镜和透射电子显微镜进行的形态学分析表明，分离的小眼细胞在形态上与原位小眼细胞几乎相同。分离的小眼细胞具有正常的视杆，在分离过程中微绒毛膜没有明显损失。目前，可以分离出含有多达六个小眼细胞的小眼，这些小眼细胞具有基本正常的结构和超微结构，包括粗视杆。分离的小眼具有裸露的A节段膜，也非常适合全细胞记录技术。
