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Michelle Giarmarco, Mark A Kanow, Ken J Lindsay, Jianhai Du, James Hurley; Confocal imaging reveals glucose uptake by photoreceptors in vivo. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1760.
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© ARVO (1962-2015); The Authors (2016-present)
Identifying the metabolic fuels used by neurons and glia in the retina is of fundamental importance for understanding retinal function and viability. In a previous study1 we showed that pyruvate kinase, the enzyme that catalyzes the final step in glycolysis, is abundant in photoreceptors (PRs) and absent from Müller glia. We hypothesize that the primary entry site for glucose in the outer retina is PRs, not Müller glia, and that PRs provide fuel to Müller glia in the form of lactate2. To test this hypothesis, we used an in vivo approach to decipher which retinal cell types take up glucose in live zebrafish larvae. We also tested the hypothesis by using immunohistochemistry (IHC) to assess the distribution of glucose transporters (GLUTs) in adult mouse retinas.(References: 1. Lindsay, K. J. et al. (2014). PNAS, 111(43), 15579–15584. 2. Hurley, J. B., Lindsay, K. J., & Du, J. (2015). J Neurosci Res, 93(7), 1079–1092.)
For in vivo glucose uptake imaging experiments, a fluorescent glucose analog, 2-NBDG, was injected into yolks of transgenic larval zebrafish expressing a red fluorescent protein marker (tdTomato) in cone PRs. Larvae were then embedded in agarose and imaged live via confocal microscopy, and colocalization between 2-NBDG and tdTomato was determined. For IHC, adult mouse eyecups were fixed in 4% paraformaldehyde and cut into 20-µm cryosections. Sections were incubated overnight with primary antibodies against CRALBP, arrestin and GLUT1, followed by incubation with fluorescent-tagged secondary antibodies. Sections were imaged via confocal microscopy.
In vivo imaging of larval zebrafish retinas revealed robust 2-NBDG uptake in cone PRs and the inner plexiform layer within minutes of injection. IHC showed that GLUT1 in mouse retina is abundant in PRs above the Müller glia apical processes, and appears to be absent from Müller glia. Because the apical processes of Müller glia partially overlap with the most inner portion of the PR cell body layer, we are developing methods to resolve definitively whether or not GLUT1 is present on the apical processes of Müller glia.
Rapid in vivo uptake of glucose into zebrafish cone photoreceptors and the presence of glucose transporters on mouse rod PR inner segments is consistent with our hypothesis that PRs and not Müller glia are the initial site of glucose entry at the outer retina.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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