June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
On-Off Direction-Selective Retinal Ganglion Cells Co-Express CART and NPAS2, a Circadian Clock Protein
Author Affiliations & Notes
  • S. Anna Sargsyan
    Emory University, Atlanta, GA
  • Christopher Hwang
    Emory University, Atlanta, GA
  • P Iuvone
    Emory University, Atlanta, GA
  • Footnotes
    Commercial Relationships S. Anna Sargsyan, None; Christopher Hwang, None; P Iuvone, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 3416. doi:
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      S. Anna Sargsyan, Christopher Hwang, P Iuvone; On-Off Direction-Selective Retinal Ganglion Cells Co-Express CART and NPAS2, a Circadian Clock Protein. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3416.

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

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Abstract

Purpose: There is a circadian rhythm of contrast sensitivity in mice, determined by optokinetic tracking, that is modulated by dopamine and neuronal PAS-domain protein 2 (NPAS2), a circadian clock component expressed in the ganglion cell layer (GCL) of the retina (Hwang et al., IOVS, ARVO Abs. 2722, 2012). On-off direction-selective retinal ganglion cells (ooDSGC) are potential mediators of NPAS2’s effect, since subtypes of these cells project directly to brain nuclei involved in the optokinetic reflex (Kay et al., J Neurosci. 31: 7753, 2011). ooDSGC can be distinguished from all other retinal ganglion cells (RGC) by a specific marker - cocaine and amphetamine regulated transcript (CART) (Kay et al., 2011). Therefore, our study aimed to investigate whether ooDSGC co-express NPAS2 and CART.

Methods: NPAS2 mutant mice, in which the second exon of the Npas2 gene has been replaced with the lacZ gene, were used to identify NPAS2 expressing cells by anti-β-galactosidase immunocytochemistry. Anti-CART immunocytochemistry was used to identify ooDSGC, and anti-Brn3a was used to identify RGC. Retinal sections of NPAS2 mutant mice were co-stained with antibodies against β-Gal and CART or β-Gal and Brn3a; nuclei were stained with DAPI. Low magnification images were obtained in tandem to reconstruct the retinal sections. ImageJ analysis software was used to perform cell counts in the GCL, as well as counts of immunoreactive RGC. Expression of Npas2 mRNA was examined in the GCL, dissected by laser capture microdissection from retinas of WT and dopamine D4 receptor-deficient (Drd4-/-) mice.

Results: β-Gal and Brn3a were co-localized in the GCL, indicating that NPAS2 is expressed in RGC. Out of all the cells in the GCL of reconstructed retinal sections, approximately 30% were positive for β-Gal. Approximately 10% of cells in the GCL were positive for CART, a selective marker for ooDSGC, and most but not all of these were β-Gal positive. In WT mice, there is a robust daily rhythm of Npas2 transcript levels in the GCL, but this rhythm was not observed in GCL of Drd4-/- mice.

Conclusions: A large majority of ooDSGC express NPAS2 as one of its circadian clock components. There is accumulating evidence that the expression of Npas2 in RGC is controlled by dopamine signaling. Thus, the circadian rhythmicity of functional outputs of ooDSGC may be modulated by dopamine via regulation of rhythmic Npas2 expression.

Keywords: 458 circadian rhythms • 688 retina  
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