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Soon Cheong, Wanyue Song, Leah Byrne, Revathi Balasubramanian, William Merigan; In vivo imaging of ChR2-RCaMP expression in retinal neurons of a transgenic blind mouse model. Journal of Vision 2014;14(15):35. doi: https://doi.org/10.1167/14.15.35.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In certain eye diseases, the selective loss of photoreceptors leads to blindness. One treatment to restore vision is to make surviving neurons light sensitive. We expressed channelrhodopsin-2 (ChR2) and red fluorescent calcium indicator (RCaMP) in retinal neurons of blind mice. Our mouse model (triple knockout, TKO; Opn4-/- Gnat1-/- Cnga3-/-; Hattar et al., 2003) lacks melanopsin as well as key molecular components in the phototransduction cascade in rods cones, and is therefore blind. Here we present preliminary images of RCaMP fluorescence imaged in the living eye.
Methods: Adult TKO and wild type B6 mice were injected intravitreally with ChR2-RCaMP with CAG promoter encapsulated in an adeno-associated virus 2 (AAV2) capsid (2μl per eye, 2.4E+13 vg/ml). RCaMP fluorescence was initially evaluated using a low-resolution scanning laser ophthalmoscope. High-resolution in vivo imaging was done with an adaptive optics scanning laser ophthalmoscope. The retinae of 5 mice were examined in greater detail using confocal microscopy. Histological analyses were carried out to determine the cell types transfected with the ChR2-RCaMP gene construct.
Results: TKO mice have normal retinal structure similar to wild type (B6) mice. RCaMP fluorescence of single cells can be detected in vivo using adaptive optics imaging. Expression of the ChR2-RCaMP is widespread and restricted to amacrine cells in both TKO and B6 mice. Future studies will investigate if activation of ChR2 leads to detectable changes in the calcium indicator fluorescence.
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