In vivo time-lapse fluorescence imaging of individual retinal ganglion cells in mice

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Abstract

We have developed a technique that permits time-lapse imaging of retinal ganglion cells (RGCs), their dendritic arbors and their axons in mammals in vivo. This technique utilizes a standard confocal laser scanning microscope, transgenic mice that express yellow fluorescent protein (YFP) in a subset of RGCs and survival anesthesia techniques. The same individual RGCs with their dendritic arbors and axons were multiply imaged in vivo in both adult and juvenile mice. Additionally, the same RGC that was imaged in vivo could then be located and imaged in fixed retinal whole mount preparations. This novel technique has many potential applications.

Introduction

A full understanding of dynamic cellular processes would benefit from the ability to observe the same cells over time rather than viewing populations of cells cross-sectionally at various time points (Lichtman and Fraser, 2001). Recently, a convergence of technological advances in molecular biology and fluorescence imaging has made it possible to image individual mammalian neurons and their processes in vivo in time-lapse fashion in both the peripheral nervous system (Walsh and Lichtman, 2003) and in the brain (Grutzendler et al., 2002, Trachtenberg et al., 2002). In the retina, time-lapse imaging of individual retinal ganglion cells (RGCs) and their dendritic processes has been possible in living zebrafish (Mumm et al., 2006, Zolessi et al., 2006). In mammals, RGC bodies have been imaged in vivo using various imaging techniques (Cordeiro et al., 2004, Engelmann and Sabel, 1999, Gray et al., 2006, Higashide et al., 2006, Sabel et al., 1997, Seeliger et al., 2005, Thanos et al., 2002). For technical reasons, in vivo imaging of individual RGCs and their dendritic arbors, which require higher resolution to image, has not been previously possible in mammals (Chalupa, 2006, Morgan et al., 2005). Time-lapse studies have provided unexpected findings regarding cellular behavior relative to what can be inferred about dynamic processes from sequential time point data (Grutzendler et al., 2002, Mumm et al., 2006, Trachtenberg et al., 2002, Walsh and Lichtman, 2003).

Thus, the ability to resolve individual RGCs and their processes in mice over time may provide new avenues of research in both normal neurophysiology and in the study of mutant or disease models. In this report, we describe a novel technique by which individual RGCs along with their dendrites and axons can be imaged in living mice in time-lapse fashion using a readily available confocal laser scanning microscope with minimal customization.

Section snippets

Animals

All protocols were approved and monitored by the Animal Care Committee of the Johns Hopkins University School of Medicine and conform to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The YFP-H line of transgenic mice which express cytoplasmic yellow fluorescent protein (YFP) in a small subset of RGCs (Feng et al., 2000) was obtained from The Jackson Laboratory (strain: B6.Cg-Tg(Thy1-YFPH)2Jrs/J; Bar Harbor, Maine). This is one line of many that express spectral

Fixed tissue imaging: retinal ganglion cells

Approximately 20 transgenic mice expressing YFP, a spectral variant of green fluorescent protein, in a small subset of RGCs (Feng et al., 2000) were utilized for these studies (The Jackson Laboratory; strain: B6.Cg-Tg(Thy1-YFPH)2Jrs/J; Bar Harbor, ME). In these mice, typically 50–100 RGCs in each retina express YFP (Fig. 2) out of an estimated 50,000 RGCs in the mouse retina (Quigley HA, unpublished data). Because the YFP is expressed cytoplasmically, it fills both cell bodies and processes

Discussion

We have been able to image individual RGCs with their axons and dendrites multiple times in a living mammal for the first time, to the best of our knowledge. Unlike zebrafish (Mumm et al., 2006, Zolessi et al., 2006), the ocular coats of pigmented transgenic mice, which express cytoplasmic spectral variants of GFP in subsets of neurons (Feng et al., 2000), prevent transscleral (posterior to the mouse cornea) imaging of RGCs using confocal laser scanning microscopy or even multiphoton microscopy

Acknowledgements

Thank you to Morton F. Goldberg, David L. Guyton, and Matthew B. Walsh for reviewing the manuscript.

Financial support: Harry A. Quigley Research Award from the Wilmer Eye Institute (MKW). NIH grants EY 02120 and 01765 (HAQ). Unrestricted support from the Leonard Wagner Trust, New York, NY (HAQ).

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Presented in part at the Wilmer Resident Association 66th Clinical Meeting, May 18, 2007, Baltimore, MD.

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