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M. R. Behrend, A. K. Ahuja, M. S. Humayun, J. D. Weiland; In vitro Retrograde Dye Loading into Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1202.
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To develop a novel retrograde loading method to load any type of fluorescent dye into retinal ganglion cells in vitro.
Neurons of the mature retina do not readily take up fluorescent indicators by bath loading of AM-ester conjugates. In vitro techniques have previously been limited to select tracer dyes manufactured in crystalline form. Here we present a more generalized in vitro method to load retinal ganglion cells with any dye in concentrated liquid form. This has applications to cell tracing and to live cell imaging with calcium sensitive dyes. A holding well is glued with cyanoacrlyate to the back of the eye encompassing the optic nerve stump of the enucleated eye. The eyecup is held in a fixture keeping the retina submerged in oxygenated culture medium at near-physiologic conditions while the optic nerve stump with dye is kept isolated above the fill level of medium. Two microliters of 5% - 10% dye solution in water was pipetted into the holding well on the optic nerve stump. Electroretinogram (ERG) and single units were recorded by placing isolated retina onto a multielectrode array recording chamber, perfused with bicabonate-buffered medium at 4 mL/min at room temperature.
Dye was found to fill ganglion cell axons, somas, and dendritic arbors following an incubation period of 1 to 3 hours. We do not observe a light-dependent photodynamic dye filling effect (Dacey, Neuron (37) 15-27, 2003) as others have for a retrograde loading procedure with the same dye in vivo with a long survival period. Labeled cell density was 300 cells/mm2 using 10,000 MW dextran-tetramethylrhodamine in mouse. Smaller dextran conjugates displayed higher labeling density as expected. In mouse 500 cells/mm2, salamander 600 cells/mm2 and in rat 800 to 1600 cells/mm2 using 3000 MW dextran-tetramethylrhodamine. Viability of the photoreceptor and inner retina cells following incubation was verified in salamander by electroretinogram response (b-wave 400 µV) to full field light flash. Ganglion cell viability following incubation was verified by recording single unit activity in response to both light flash and biphasic current pulses 50 µA, 400 µs per phase.
We have demonstrated the efficacy and tissue viability for a new retrograde dye loading method for retinal ganglion cells. This method is compatible with calcium sensitive dyes capable of live cell imaging.
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