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Z.–H. Pan, A. Bi, Y.–P. Ma, E. Olshevskaya, A.M. Dizhoor; Functional Expression of a Directly Light–Gated Membrane Channel in Mammalian Retinal Neurons: A Potential Strategy for Restoring Light Sensitivity to the Retina After Photoreceptor Degeneration . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4631.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Congenital retinal degenerative diseases, such as retinitis pigmentosa, result in death of photoreceptor cells and a complete loss of retinal responses to light. To explore the feasibility of converting inner retinal neurons to photosensitive cells as a possible strategy for restoration of light sensitivity in the retina, we investigated the functional expression in retinal neurons a directly light–gated cation–selective membrane channel, channelrhodopsin–2, recently reported by Nagel et al. (2003). Methods: A Chop2–GFP chimera was made by tagging GFP at the C–terminal of the channelopsin–2 (Chop2). The Chop2–GFP was transfected into HEK cells under control of a CMV promoter. A viral construct was made by subcloning the Chop2–GFP into an AAV–2 viral cassette containing a CAG promoter. The viral vectors were injected into the eye of new born rats. The expression of Chop2–GFP was examined by GFP fluorescence in retinal whole–mounts or slice sections. The function of the Chop2–GFP was assessed by whole–cell patch clamp recordings. Results: Bright GFP fluorescence was detected within 18–24 hrs in HEK cells after the transfection. The fluorescence was localized predominantly to the plasma membrane. The preserve of the function of the Chop2–GFP chimera was confirmed by patch–clamp recordings. Substantial light–gated currents were also observed in the Chop2–GFP–expressing HEK cells without adding the exogenous all–trans retinal, indicating that a significant number of functional Chop2–GFP channels were formed in HEK cells using only endogenous precursor for the chromophore group. Three to four weeks after the injection, GFP fluorescence was observed in the retinal neurons of the injected eyes. Bright GFP–fluorescence was observed in many ganglion cells and horizontal cells, some amacrine cells, and, occasionally, bipolar cells for at least 10 weeks following injection. The Chop2–GFP–expressing retinal neurons exhibited robust membrane depolarization in response to light stimulation and did not require an exogenous source of all–trans retinal. Conclusions: We demonstrate the functional expression of a directly light–gated membrane channel, channelrhodopsin–2, in rat retinal neurons in vivo. Our results suggest that expression of light–gated membrane channels in second– or third–order retinal neurons is a potential strategy for restoration of light perception after degeneration of photoreceptors.
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