Daniele Lauren L, Lillo Concepcion, Lyubarsky Arkady L, Nikonov Sergei S, Philp Nancy, Mears Alan J, Swaroop Anand, Williams David S, Pugh Edward N
F. M. Kirby Center for Molecular Ophthalmology, Department of Ophthalmology, School of Medicine, University of Pennsylvania, Philadelphia, 19104, USA..
Invest Ophthalmol Vis Sci. 2005 Jun;46(6):2156-67. doi: 10.1167/iovs.04-1427.
To test the hypothesis that Nrl(-)(/)(-) photoreceptors are cones, by comparing them with WT rods and cones using morphological, molecular, histochemical, and electrophysiological criteria.
The photoreceptor layer of fixed retinal tissue of 4- to 6-week-old mice was examined in plastic sections by electron microscopy, and by confocal microscopy in frozen sections immunolabeled for the mouse UV-cone pigment and colabeled with PNA. Quantitative immunoblot analysis was used to determine the levels of expression of key cone-specific proteins. Single- and paired-flash methods were used to extract the spectral sensitivity, kinetics, and amplification of the a-wave of the ERG.
Outer segments of Nrl(-/-) photoreceptors ( approximately 7 mum) are shorter than those of wild-type (WT) rods ( approximately 25 mum) and cones ( approximately 15 mum); but, like WT cones, they have 25 or more basal discs open to the extracellular space, extracellular matrix sheaths stained by PNA, chromatin "clumping" in their nuclei, and mitochondria two times shorter than rods. Nrl(-/-) photoreceptors express the mouse UV cone pigment, cone transducin, and cone arrestin in amounts expected, given the relative size and density of cones in the two retinas. The ERG a-wave was used to assay the properties of the photocurrent response. The sensitivity of the Nrl(-/-) a-wave is at its maximum at 360 nm, with a secondary mode at 510 nm having approximately one-tenth the maximum sensitivity. These wavelengths are the lambda(max) of the two mouse cone pigments. The time to peak of the dim-flash photocurrent response was approximately 50 ms, more than two times faster than that of rods.
Many morphological, molecular, and electrophysiological features of the Nrl(-/-) photoreceptors are cone-like, and strongly distinguish these cells from rods. This retina provides a model for the investigation of cone function and cone-specific genetic disease.
通过使用形态学、分子学、组织化学和电生理学标准，将Nrl基因敲除（Nrl(-)(/)(-)）的光感受器与野生型（WT）视杆细胞和视锥细胞进行比较，以验证Nrl(-)(/)(-)光感受器是视锥细胞这一假设。
对4至6周龄小鼠固定视网膜组织的光感受器层进行塑料切片，通过电子显微镜检查，并在冷冻切片中进行共聚焦显微镜检查，这些冷冻切片用小鼠紫外视锥色素进行免疫标记，并与花生凝集素（PNA）进行共标记。采用定量免疫印迹分析来确定关键视锥细胞特异性蛋白的表达水平。使用单闪光和双闪光方法来提取视网膜电图（ERG）a波的光谱敏感性、动力学和放大倍数。
Nrl基因敲除的光感受器的外段（约7μm）比野生型视杆细胞（约25μm）和视锥细胞（约15μm）的外段短；但是，与野生型视锥细胞一样，它们有25个或更多向细胞外空间开放的基底膜盘，有被PNA染色的细胞外基质鞘，细胞核中的染色质“结块”，以及线粒体比视杆细胞短两倍。考虑到两个视网膜中视锥细胞的相对大小和密度，Nrl(-/-)光感受器表达的小鼠紫外视锥色素、视锥转导素和视锥抑制蛋白的量符合预期。ERG a波用于检测光电流反应的特性。Nrl(-/-) a波的敏感性在360nm处达到最大值，在510nm处有一个次要峰值，其最大敏感性约为最大值的十分之一。这些波长是两种小鼠视锥色素的最大吸收波长（λmax）。弱闪光光电流反应的峰值时间约为50毫秒，比视杆细胞快两倍多。
Nrl(-/-)光感受器的许多形态学、分子学和电生理学特征类似视锥细胞，并将这些细胞与视杆细胞强烈区分开来。该视网膜为研究视锥细胞功能和视锥细胞特异性遗传疾病提供了一个模型。
