Cortical Responses Driven by an Engineered Light Activated Glutamate Receptor in Models of Inherited Blinding Diseases

Abstract

Purpose: : A potential therapy for late stage blinding diseases, where the majority of photoreceptors have degenerated, is to confer light sensitivity on surviving retinal interneurons. We delivered an engineered light sensitive protein, the light activated glutamate receptor (LiGluR), to retinal ganglion cells in an animal model of retinal degeneration (rd-1 mice). LiGluR has been shown to control single neuron activity with millisecond resolution in cultured hippocampal cells (Szobota et al. 2007). To begin to understand how the visual cortex interprets engineered visual input, we have recorded LiGluR mediated population responses in primary visual areas to full field visual stimulation.

Methods: : LiGLuR was cloned into an AAV-2 vector backbone. AAV-2 pseudotyped virus was packaged by triple transfection and purified to generate high-titer vector (10¹²-10¹³ vg/mL). The virus was injected into the vitreous of 6-month-old rd-1 mice and allowed at least 3 weeks to reach peak expression. Animals were subsequently intravitreally injected with the photo-switchable compound, MAG (maleimide-azobenzene-glutamate). At 6, 24, or 48 hours post-MAG-injection, V1 cell population responses were recorded (VEPs) in response to wavelengths ranging from 320nm-480nm. Following VEP recording, retinas were prepared for immunohistochemical localization of iGluR6 to determine the LiGluR expression profile.

Results: : LiGluR expression in retinal ganglion cells of rd-1 mice drives robust population responses in V1. The VEP response amplitude peaks at 380nm but remains robust in the visible range. We observed clear visually evoked responses at all post-injection time points, with a maximum response amplitude at 24hrs. Cortical responses to LiGluR mediated light input were at a minimum, three orders of magnitude higher than control rd-1 mice expressing the light activated channel, channelrhodopsin, in the RGCs.

Conclusions: : Our data show that the engineered photoswitch, LiGLuR, when expressed in the retinal ganglion cells, can drive cortical responses to full field light flashes in "blind" animals. Our data suggest that LiGluR drives VEPs more effectively than any light sensitive channel tested to this point. This suggests that LiGluR could be a good candidate for the restoration of light sensitivity in late stages of retinal degeneration.

Keywords: gene transfer/gene therapy • retina • retinal degenerations: cell biology