March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
Assessment Of Two Cre-lox Reporters Of Melanopsin Expression
Author Affiliations & Notes
  • Lauren E. Quattrochi
    Molecular Pharmacology and Physiology,
    Department of Neuroscience,
    Brown University, Providence, Rhode Island
  • Maureen E. Estevez
    Department of Neuroscience,
    Brown University, Providence, Rhode Island
  • David M. Berson
    Department of Neuroscience,
    Brown University, Providence, Rhode Island
  • Footnotes
    Commercial Relationships  Lauren E. Quattrochi, None; Maureen E. Estevez, None; David M. Berson, None
  • Footnotes
    Support  R01 EY012793
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 4341. doi:
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      Lauren E. Quattrochi, Maureen E. Estevez, David M. Berson; Assessment Of Two Cre-lox Reporters Of Melanopsin Expression. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4341.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Mice with cre knocked into the melanopsin locus are a powerful tool for identifying intrinsically photosensitive retinal ganglion cells (ipRGCs; Ecker et al. 2010, Neuron, 2008). We assessed their utility and reliability when crossed with two lines of reporter mice, seeking to understand mismatched expression of fluorescent protein and melanopsin immunostaining and whether more sensitive reporter mice would reveal new ipRGC types.

Methods: : We crossed Opn4cre/cre mice (Ecker et al. 2010) with two floxed-stop reporter mice: "Z/EG" (Jax#003920) or "tdTom" (Jax#0079095); offspring should express GFP or td-Tomato, respectively, in cre-expressing cells. Fluorescent cells were injected with Lucifer Yellow and morphologically identified. Melanopsin was visualized with anti-melanopsin antibody (UF006) enhanced by tyramide signal amplification (TSA).

Results: : In Opn4cre/+;Z/EG +/- mice, though M4 and M5 types expressed GFP and were intrinsically photosensitive, standard immunohistochemical methods failed to detect melanopsin (Ecker et al. 2010). Here, however, TSA revealed the opsin in the great majority of M4 cells and at least some M5 cells identified by dye-filling. Roughly 75% of the largest GFP+ cells (presumptive M4s) were melanopsin-positive after TSA. Among presumptive M5 cells (GFP+ cells too small to be M4 cells and too weakly melanopsin-immunolabeled to be M1, M2, or M3 cells), a third were immunopositive by TSA. The Z/EG reporter does not label all ipRGCs: 20-25% of immuno-detected M1, M2 and M4 cells lacked GFP. In the tdTom reporter (Opn4cre/+;tdTom +/- ), at least twice as many ganglion cells were fluorescent as in Z/EG mice, and most lacked detectable melanopsin immunolabeling. Dye-filled tdTom+ cells were far more diverse than GFP+ cells, including ON-OFF DS cells and other types never labeled in the Z/EG reporter. Many td-Tom+ cells lacked intrinsic photosensitivity, whereas this was almost never true for GFP+ cells.

Conclusions: : M4 and M5 cells are true melanopsin-expressing ipRGCs. The Opn4cre/+;Z/EG +/- mouse is the best available reporter of melanopsin expression in ganglion cells, though it does fail to mark a minority of melanopsin cells and may label a few cells that do not express the opsin. By contrast, the Opn4cre/+;tdTom +/- reporter is excessively sensitive, labeling many cells that do not express functionally meaningful levels of melanopsin.

Keywords: microscopy: light/fluorescence/immunohistochemistry • ganglion cells • electrophysiology: non-clinical 

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