April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
DNA Microarray Analysis of Melanopsin-Containing Retinal Ganglion Cells
Author Affiliations & Notes
  • L. D. Squires
    Univ. of Washington School of Medicine, Seattle, Washington
  • R. L. Brown
    VCAPP, Washington State University, Pullman, Washington
  • Footnotes
    Commercial Relationships  L.D. Squires, None; R.L. Brown, None.
  • Footnotes
    Support  UW MSRTP Program (LDS); NIH Grant MH67094 (RLB)
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 5036. doi:
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      L. D. Squires, R. L. Brown; DNA Microarray Analysis of Melanopsin-Containing Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5036.

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

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Purpose: : The circadian pacemaker in the mammalian SCN is synchronized with the solar cycle by direct synaptic input from a small subset of retinal ganglion cells (RGCs), which contain the novel photopigment, melanopsin, and are intrinsically photosensitive. Due to the scarcity of these cells (~ 2000/retina), it has proven difficult to elucidate the melanopsin-based phototransduction cascade. The recent production of a mouse strain expressing GFP in the melanopsin-containing (m)RGCs (the gift of S. Hattar, JHU) has enabled us to use florescence-activated cell sorting (FACS) to generate highly-enriched cell populations for differential gene expression profiling.

Methods: : Transgenic mice were constructed that express GFP in the mRGCs. Retinas from transgenic and control mice were dissociated by treatment with papain, yielding a suspension of single cells. FACS enabled us to obtain > 4, 000 GFP-positive cells from twenty retinas. Control RGCs were specifically labeled with the Thy-1 marker and purified by FACS. Total RNA was isolated from both cell populations, converted to cDNA, and amplified in a linear fashion. This cDNA was applied to an Illumina mouse-6 v1.1 array, containing probes for > 22, 000 transcripts; hybridization results were analyzed using "Genesifter" (VizXLabs).

Results: : As expected, the melanopsin transcript (Opn 4) was enriched in mRGCs by 31-fold over whole retina, and 17-fold over the total RGC population. The transcript encoding the pituitary adenylyl cyclase activating peptide (PACAP), another marker for mRGCs, was enriched 38-fold over whole retina and 14-fold over Thy-1+ RGCs. In contrast, the relative abundance of standard photoreceptor markers was reduced by 10- to 25-fold in the mRGCs. Furthermore, TRPC7, previously reported to be enriched in mRGCs, was enriched 9-fold over total retina, and G14 was enriched 11-fold over total retina (10-fold over RGCs).

Conclusions: : Elucidation of the melanopsin-based signaling pathway is crucial to the understanding of circadian photoentrainment. Our study has identified several candidate proteins as potential components of the phototranduction cascade; future functional characterization studies will be required to confirm their role.

Keywords: ganglion cells • gene microarray • circadian rhythms 

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