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Satchidananda Panda, Sheena R. Keding, Hiep D. Le, Daniel Gibbs, James Fitzpatrick, Megumi Hatori; Comprehensive Analysis Of The Central Projections Of Melanopsin-expressing Retinal Ganglion Cell In Mice. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3460.
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© ARVO (1962-2015); The Authors (2016-present)
Melanopsin (Opn4) and melanopsin expressing retinal ganglion cells (mRGCs) have now been implicated in several photoresponses. These mRGCs project their axons to the suprachiasmatic nucleus (SCN) and a few other brain regions that directly or indirectly regulate all non-image forming visual processes including circadian photoentrainment, pupil constriction, pineal melatonin regulation and light regulation of activity/rest. Identifying the full complement of mRGCs and their central projection is critical to understanding the cellular basis of melanopsin function. However, the existing methods to mark melanopsin cells and map their projections are insufficient. We have developed novel methods to comprehensively map mRGC projections.
We bred Opn4Cre mouse to Z/EG or Z/AP mice, which allows Cre-dependent expression of green fluorescent protein (GFP) or human alkaline phosphatase (AP) from a β-actin promoter. We also used Adeno-associated virus serotype 2 (AAV2) expressing a green or red fluorescent protein in Cre-dependent manner in intravitreal injections to Opn4Cre mice to specifically label the mRGCs. Flat mount retina, retina sections, and brain sections were stained for the respective reporters.
In the retina of Opn4Cre/+;Z/EG mice, GFP expressing cells were mostly found in the retinal ganglion cell (RGC) sub-layer, and these cells had extensive dendritic arborization characteristic of the mRGCs. In Opn4Cre/+;Z/AP mice, the mRGCs strongly innervate the SCN. Additionally, mRGCs axon termini also sparsely innervate various other hypothalamic regions. Surprisingly, the AP staining also revealed extensive projection of the mRGCs in the lateral geniculate complex which is involved in image-forming vision. However, in adult mice, it was reported that there was a chance of insufficient labeling of Z/EG and Z/AP lines probably due to the methylation of the β -actin promoter used for these lines. Therefore we had intravitreally injected AAV2 viruses that express mCherry or EGFP from a different promoter in Cre-dependent manner to adult mice. This attempt resulted in reproducible success in transducing vast majority of mRGCs only in the injected retina than the retina of Opn4Cre/+;Z/EG mice.
We found that the projection patterns of mRGCs were much more extensive than previously reported. We also developed the novel viral strategy to comprehensively map mRGC projections. Such strategy can now be applied to any mouse expressing Cre in the retina.
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