Optic neuropathies, such as glaucoma, lead to retinal ganglion cell (RGC) death. Transgenic mouse strains that express fluorescent proteins under the control of the Thy1 promoter have permitted single RGC imaging. Specifically, in one strain of mice expressing yellow fluorescent protein (Thy1-YFP), fluorescence is expressed in only 0.2% of RGCs. This reduced expression allows visualization of the full dendritic arbour of YFP-expressing RGCs, facilitating the investigation of structural changes. As susceptibility amongst RGCs varies with morphology and subtype, labelling methods should ideally non-discriminately label RGCs to accurately determine the effects of experimental glaucoma. This study therefore sought to determine morphological subtypes of RGCs in the Thy1-YFP mouse strain. Retinas from Thy1-YFP mice were imaged ex vivo with fluorescence microscopy. With Sholl analysis, a technique for quantifying the morphology of individual neurons, the dendritic field (DF), area under the curve (AUC), normalized AUC (Nav), peak number of intersections (PNI), and skew for single RGCs were computed. The distance of the RGC from the optic nerve head (dONH) was also measured. These morphological parameters were inputted into a multivariate cluster analysis to determine the optimal number of clusters to group all RGCs analyzed, which were then grouped into "Small", "Medium", and "Large" sized cluster groups according to increasing DF size. A total of 178 RGCs from 10 retinas of 8 mice were analyzed from which the cluster analysis identified 13 clusters. Eighty-eight (49%), 77 (43.2%), and 13 (7.3%) RGCs were grouped into small, medium and large clusters, respectively. Clusters 1-6 had small DFs. Clusters 1 and 3 had the lowest AUC and Nav. Clusters 2, 3, and 5 had asymmetric DFs while Clusters 3, 5, and 6 were distal to the ONH. Clusters 7-11 had medium DFs; of these, Clusters 7 and 10 had the lowest AUC, Clusters 8 and 10 had the highest skew, and Clusters 7 and 11 were closest to the ONH. Clusters 12 and 13 had large DFs. Both had low skew and high AUC. High PNI and dONH distinguished Cluster 12 from Cluster 13. We present the largest study to date examining YFP expression in RGCs of transgenic Thy1-YFP mice. Among the 13 clusters, there was a wide range of morphological features with further variation within size categories. Our findings support the notion that YFP is expressed non-discriminatingly in RGCs of Thy1-YFP transgenic mice and this strain is a valuable tool for studies of experimental optic neuropathies.

中文翻译：

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选择性表达黄色荧光蛋白的鼠视网膜神经节细胞的形态学多元聚类分析。

视神经病变（例如青光眼）会导致视网膜神经节细胞（RGC）死亡。在Thy1启动子控制下表达荧光蛋白的转基因小鼠品系已允许进行单个RGC成像。具体而言，在一只表达黄色荧光蛋白（Thy1-YFP）的小鼠品系中，荧光仅在0.2％的RGC中表达。这种减少的表达允许可视化表达YFP的RGC的完整树突状乔木，从而便于研究结构变化。由于RGC之间的易感性随形态和亚型的不同而变化，因此理想地，标记方法应无差别地标记RGC，以准确确定实验性青光眼的作用。因此，本研究试图确定Thy1-YFP小鼠品系中RGC的形态亚型。Thy1-YFP小鼠的视网膜通过荧光显微镜离体成像。通过Sholl分析，可以计算出用于量化单个RGC的单个神经元的形态，树突场（DF），曲线下面积（AUC），归一化AUC（Nav），相交峰数（PNI）和偏斜的技术。 。还测量了RGC与视神经头的距离（dONH）。将这些形态参数输入到多元聚类分析中，以确定将所有RGC分组的最佳聚类数，然后根据DF大小的增加将其分为“小”，“中”和“大”大小的聚类组。分析了来自8只小鼠的10个视网膜的178个RGC，通过聚类分析确定了13个聚类。八十八（49％），77（43.2％）和13（7。3％）RGC分别分为小集群，中集群和大集群。集群1-6的DF较小。群集1和3的AUC和Nav最低。群集2、3和5具有不对称DF，而群集3、5和6位于ONH的远端。集群7-11有中等DF。其中，群集7和10的AUC最低，群集8和10的偏斜最高，群集7和11最接近ONH。群集12和13具有较大的DF。两者都具有低偏斜和高AUC。高PNI和dONH将簇12与簇13区分开。我们提出了迄今为止最大的研究，目的是检验转基因Thy1-YFP小鼠RGC中YFP的表达。在13个簇中，形态特征范围广泛，并且大小类别之间存在进一步的差异。