Abstract
Purpose :
Ectopic expression of ChannelRhodopsin2 in retinal ganglion cells was shown to restore vision in blind rd1 mice. However, concerns on phototoxicity were raised by the required high excitation threshold in the blue wavelength range. In this study, we investigated the use of ChrimsonR (ChrR), a red shifted opsin, as radiation safety limits are much higher in the red light range. We compared further the functional efficacy of ChrR and the construct ChrimsonR-TdTomato (ChrR-TdT) in two degenerative models: rd1 and P23H.
Methods :
rd1 mice and P23H rats were injected intravitreally with AAV2.7m8 vector encoding ChrR-Tdt and ChrR under the CAG promoter. Expression of TdT was examined by its fluorescence under classic and confocal microscopy. Multielectrode array (MEA) recordings were performed ex vivo on flat-mounted retina in presence of synaptic blockers to assess photoactivation of ChrR-expressing retinal ganglion cells at least a month after injection.
Results :
ChrR-tdT expression was widely distributed across the RGC layer in the rd1 retina. The fluorescence extended in RGC cell bodies and their dendritic arbors. For MEA recordings, retinal pieces were selected close to the optic nerve where transfection was maximal. For rd1 experiments, retinal ganglion cell responses were measured in 6 out of 8 retinas with ChrR-TdT whereas only 4 retinas out of 8 were active with ChrR. The activity spectrum matched that of ChrR. In the red wavelength range, light level to elicit spiking was below radiation safety limits. Light thresholds were similar with ChrR and ChrRTdT but spiking frequencies were greater in ChrR-TdT expressing cells. Recording in P23H rats confirmed retinal ganglion cell photoactivity following ChR-TdT delivery.
Conclusions :
This study demonstrated the potential of ChrR for the reactivation of retinal ganglion cells in a blind retina. The data suggested that ChrR-TdT was more potent than ChrR and paved the way for further investigation of ChrR-TdT expression and function in the primate retina (see posters Gauvain et al. and Chaffiol et al.).
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.