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Sandhya S. Rao, YiHui Hu, MingTing Tian, Shruti Mistry, Lauren M. Luhrs, John E. Donello, Karen M. Kedzie, Daniel W. Gil; Localization Of α2A Adrenergic Receptors In Retina Of HATag-α2A Knock-In Mouse Model. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2059.
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© ARVO (1962-2015); The Authors (2016-present)
Distribution and localization of α2A receptors in eye including the retina have been challenging due to lack of receptor selective pharmacophores & antibodies. Recombinant gene targeting strategy was used to generate a knock-in mouse line that expresses an N-terminal heamagglutinin epitope tagged-α2A receptor driven by endogenous α2A AR gene locus (collaboration:Dr. L Limbard,Vanderbilt Univ Medical Center,TN) to localize retinal α2A receptors.
Immunohistochemistry studies utilizing HAtag antibody and retinal markers were performed to evaluate distribution & localization of α2A AR in mouse retina. All animal expts were done with protocols approved by Allergan Institutional Animal Care & Use Committee. Transverse retinal sections were obtained from HAtag-α2A KI (Homo&WT) mice 4% para fixed eye cups. Primary antibody incubations were @ 40C overnight with HA.11 Clone 16B12 Monoclonal antibody(1:500,Covance) alone/Glutamine Synthetase(1:10000,AbCam), PKCa(1:250,Novus)/Calbindin-D(1:2500,Millipore). After incubations in secondary antibodies (Dylight488/649,Jackson IR) slides were coverslipped using DAPI Fluoromount-G(SouthernBiotech). Images were obtained by laser scanning Confocal Microscopy (Zeiss710,Zen2009).
IHC results indicate punctate labeling by HAtag antibody (Ab) in outer plexiform layer. HAtag Ab also labels cell membranes of some cells of inner nuclear layer(INL). Lower levels of expression obtained in Inner Plexiform Layer(IPL) & Retinal Ganglion Cell layer(RGC). Double labeling results indicate HAtag Ab does not co-localize with PKCa(rod bipolar cells) marker or Glutamine Synthetase marker (Muller Cells). Double labeling data indicates co-localization of HA tag Ab with horizontal cell marker(Calbindin-D). HAtag Ab labels membrane bound α2A receptors in horizontal cell bodies in INL & synaptic terminals of horizontal cells in OPL.
Immunostaining data indicates α2A receptors are expressed in several layers of retina. Punctate staining obtained in OPL indicate receptors are expressed in synaptic terminals, an expected location for neuromodulatory & presynaptic α2A receptors. Novel findings indicate α2A receptors are localized on horizontal cells of HAtag-α2A knock-in mouse retina. Low levels of expression were also found in INL, IPL & RGC of HAtag-α2A knock-in mouse retina
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