Abstract
Purpose: :
By expressing a light-gated cation channel, channelrhodopsin-2 (ChR2), in inner retinal neurons, we have previously demonstrated the ability of restoring ON responses in the retina after the death of rod and cone photoreceptors. In this study, we investigated the feasibility of restoring OFF responses to the retina by expressing halorhodopsin (HaloR), a light-driven chloride pump, in inner retinal neurons. In addition, we examined the co-expression of HaloR and ChR2 and studied the light response properties of the co-infected cells.
Methods: :
Adeno-associated virus serotype 2 (AAV2) vectors carrying fusion constructs of HaloR-mCherry and Chop2-GFP with CMV promoter were injected into the intravitreal space of the eyes of adult C57BL/6J and C3H/HeJ (rd1/rd1) mice. The expression of HaloR and ChR2 was visualized by mCherry and GFP fluorescence in retinal whole-mounts or vertical sections. The light response properties of HaloR-expressing inner retinal neurons were studied by whole-cell patch-clamp recordings on acutely isolated cells and cells in retinal slices. The HaloR- and ChR2-mediated spike activities of retinal ganglion cells were assessed by multi-electrode array recordings of whole-mount retinas.
Results: :
Functional expression of HaloR was achieved in inner retinal neurons in vivo. HaloR-expressing retinal ganglion cells responded to light with a rapid membrane hyperpolarization or suppression of spike activities. At the light offset, their membrane potential exhibited a rapid rebound overshoot with robust spike firing. Both sustained and transient OFF-responses mediated by HaloR were observed. Co-expression of ChR2/HaloR in retinal ganglion cells could produce ON, OFF, and even ON-OFF responses dependent on the light wavelengths.
Conclusions: :
Expression of HaloR can effectively restore OFF responses in inner retinal neurons of a mouse with retinal degeneration. Targeted expression of ChR2 and HaloR to ON and OFF inner retinal neurons, respectively, or co-expression of ChR2/HaloR in retinal ganglion cells are the possible approaches to restore both ON and OFF light responses in the retina after the death of rod and cone photoreceptors.
Keywords: gene transfer/gene therapy • retinal degenerations: hereditary • electrophysiology: non-clinical