Semi-automated, quantitative analysis of retinal ganglion cell morphology in mice selectively expressing yellow fluorescent protein.
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.
Glaucoma Center of Excellence, Wilmer Ophthalmological Institute, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21287-9205, USA. eoglesb1@jhmi.edu
Ericka
E
Oglesby
Harry A
HA
Quigley
Donald J
DJ
Zack
Frances E
FE
Cone
Matthew R
MR
Steinhart
Jing
J
Tian
Mary E
ME
Pease
Giedrius
G
Kalesnykas
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D002452:Cell Count; D002460:Cell Line; D005456:Fluorescent Dyes / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic; D012165:Retinal Ganglion Cells / Q000166:cytology* / Q000378:metabolism; D018800:Thy-1 Antigens
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D018800:Thy-1 Antigens; C054562:yellow fluorescent protein, Bacteria