Semi-automated, quantitative analysis of retinal ganglion cell morphology in mice selectively expressing yellow fluorescent protein

Abstract

The development of transgenic mouse lines that selectively label a subset of neurons provides unique opportunities to study detailed neuronal morphology and morphological changes under experimental conditions. In the present study, a mouse line in which a small number of retinal ganglion cells (RGCs) express yellow fluorescent protein (YFP) under control of the Thy-1 promoter was used (Feng et al., 2000). We characterized the number, distribution by retinal region and eccentricity of YFP-labeled RGCs using fluorescence microscopy and Stereo Investigator software (MicroBrightField, VT, USA). Then, we captured images of 4–6 YFP-expressing RGCs from each of 8 retinal regions by confocal microscopy, producing 3-dimensional and flattened data sets. A new semi-automated method to quantify the soma size, dendritic length and dendritic arbor complexity was developed using MetaMorph software (Molecular Devices, PA, USA). Our results show that YFP is expressed in 0.2% of all RGCs. Expression of YFP was not significantly different in central versus peripheral retina, but there were higher number of YFP-expressing RGCs in the temporal quadrant than in the nasal. By confocal-based analysis, 58% of RGCs expressing YFP did so at a high level, with the remainder distributed in decreasing levels of brightness. Variability in detailed morphometric parameters was as great between two fellow retinas as in retinas from different mice. The analytic methods developed for this selective YFP-expressing RGC model permit quantitative comparisons of parameters relevant to neuronal injury.

Introduction

Glaucoma is the second leading cause of blindness worldwide (Quigley and Broman, 2006) and its principal pathological feature is the death of RGCs (Quigley et al., 1981). Apoptosis is a final common pathway for RGC death both in human and experimental models of glaucoma (Quigley et al., 1995; Kerrigan et al., 1997). There is some evidence that, prior to cell death, RGCs undergo decreases in either soma size and dendritic arborization (Weber et al., 1998). In the spontaneous glaucoma exhibited by DBA/2J mice and experimental glaucoma in rats, there is temporary persistence of RGC bodies with an altered dendritic phenotype after axon loss (Jakobs et al., 2005; Buckingham et al., 2008; Son et al., 2010; Soto et al., 2011). Similarly, some neurons undergo change in structure prior to death in Alzheimer’s, Parkinson’s and Huntington’s diseases (Kisiswa et al., 2010).

Further support for alterations in RGCs prior to their final somal death process has been detected by functional testing after experimental injury. Pattern-evoked electroretinography (ERG) (Johnson et al., 1989) and detailed functional recordings (Weber and Harman, 2005) point to altered RGC responses in monkey models of glaucoma. ERG changes in waveforms thought to be derived from RGC layer cells are seen in rodent glaucoma (Holcombe et al., 2008; Kong et al., 2009; Porciatti and Ventura, 2009). If either structural or functional change in RGCs prior to cell death were measurable in human eyes, these parameters could serve as early indications of neuronal injury, potentially at a reversible stage. Such a biomarker would be useful in shortening the length of human clinical trials for neuroprotective agents.

The recent development of a transgenic mouse in which a small number of RGCs express yellow fluorescent protein (YFP) provides unique opportunities to study detailed structural change. In transgenic mice in which every RGC is labeled (Feng et al., 2000), individual RGC dendritic and axonal morphology cannot be resolved. By contrast, RGCs in the retina of selectively expressing YFP mice are sparsely labeled, so the individual cell characteristics of dendritic, somal, and axonal anatomy can be examined in detail and quantified. Our laboratory and others found it possible to image the same RGC over time in living mice of this strain (Walsh and Quigley, 2008; Leung et al., 2008). A recent report showed serial, in vivo observations of YFP-selective expressing RGCs in mice after optic nerve crush injury (Leung et al., 2011), suggesting dendritic shrinkage prior to death. By contrast, another in vivo study using optical coherence tomography suggested that the retina thickened prior to RGC death in mice after optic nerve crush (Gabriele et al., 2011).

To study changes in RGC morphology after experimental glaucoma or nerve injury in the YFP-selective expression mice, we developed methods for detailed analysis of neuronal structure in whole-mounted retina. These methods can specify the phenotypic alterations that seem likely to be occurring between the time of significant axonal injury and somal death. While in vivo imaging has the advantage of following individual RGCs over time, it can view only a small number of cells. The changes in corneal clarity in chronic experimental mouse glaucoma often reduce resolution of detailed measurement in vivo. Though the morphology of individual cells can be followed to some extent in the living eye, a detailed, 3-dimensional analysis cannot be performed as accurately in vivo as it can be with confocal imaging of fixed tissues or of retinal explants.

Past approaches to quantify neuronal morphology in neural tissues included the histological Golgi method, immunohistochemical delineation of the cytoskeleton (Jakobs et al., 2005), intracellular single-cell dye injections (Tauchi and Masland, 1985), or biolistic delivery of fluorescent dyes (Gan et al., 2000). In addition to mice expressing YFP in a select number of RGCs, Nathans and co-workers have produced transgenic mouse lines exhibiting sparse labeling of RGCs, amacrine cells and other retinal neurons (Badea et al., 2003, Badea et al., 2009). Quantitative evaluation of neuronal morphology has recently been improved by objective, semi-automated methods (Gensel et al., 2010) that provide quantitative data on axon length and dendrite branching complexity, also known as Sholl analysis (Sholl, 1953).

In vitro study of whole-mounted retinas allows detailed, quantitative observations on RGC dendritic, somal, and axonal components, and can be paired with immunolabeling of important additional biomarkers and non-RGC cells that may be specifically related to injury and disease. We and others have developed inducible models of chronic glaucoma in mice (Sappington et al., 2010; Cone et al., 2010) that produce RGC death and may permit study of initial changes in RGC morphology. By detailed analysis of the earliest phase of injury, it is possible that features can be detected that would be usable clinically to identify a reversible stage of RGC injury. This report details the characteristics of RGCs that selectively express YFP in mice, analyzed by newly developed morphological methods and comparing data from different regions and eccentricities of the retina. A companion report will give the detailed changes from YFP mice after nerve crush and glaucoma.