Abstract
Purpose:
The loss of photoreceptor cells caused by retinal degeneration leads to partial or complete blindness such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). As a strategy to restore the function in retinal degenerative diseases, several researchers suggested the functional optogenes such as channelrhodopsin-2 (ChR2) or Halorhodopsin (NpHR), microbial-type rhodopsin, which could play a role as a light sensor and could produce action potential firings in retina. Here, to employ the complete set of retinal circuits for ON/OFF signal, ChR2 and NpHR were targeted to ON-bipolar cells and OFF-bipolar cells, respectively, by target-specific promoter (Grm6 and ChX10 promoter) and Cre-loxp system.
Methods:
To transduce optogenes into the retinas, we used AAV8-Y733F mutant vectors (AAV8m) that reported the improvement of the cellular transduction. And, we applied viral cassettes, AAV8m-Grm6-ChR2-YFP-Cre and AAV8m-Chx10-loxp-NpHR-mCherry-loxp. The viral vectors were injected into subretinal space of eyes. The expression of ChR2 and NpHR was examined in whole mount retina and vertical section using confocal microscope.
Results:
One month after viral injection, ChR2, green fluorescence and NpHR, red fluorescence were observed in the whole mount retina. Especially, in the retinal vertical section, ChR2 and NpHR was expressed by target-specific promoters in the layer of bipolar cells. And in the PKC-alpha-positive cells, the ON bipolar cells, ChR2 was expressed, whereas NpHR was not expressed by Cre-loxp system.
Conclusions:
In this study, we have showed that ChR2 and NpHR were expressed the ON-bipolar cells and OFF-bipolar cells, respectively, using Cre-loxp system. Therefore, we suggest that our systems may realize the light-sensitive inner retinal neurons in photoreceptor-deficient retinas, thereby activating the ON and OFF pathways with opposite polarity.
Keywords: 688 retina •
435 bipolar cells