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, 81 (4), 800-13

Restoring Visual Function to Blind Mice With a Photoswitch That Exploits Electrophysiological Remodeling of Retinal Ganglion Cells


Restoring Visual Function to Blind Mice With a Photoswitch That Exploits Electrophysiological Remodeling of Retinal Ganglion Cells

Ivan Tochitsky et al. Neuron.


Retinitis pigmentosa (RP) and age-related macular degeneration (AMD) are blinding diseases caused by the degeneration of rods and cones, leaving the remainder of the visual system unable to respond to light. Here, we report a chemical photoswitch named DENAQ that restores retinal responses to white light of intensity similar to ordinary daylight. A single intraocular injection of DENAQ photosensitizes the blind retina for days, restoring electrophysiological and behavioral responses with no toxicity. Experiments on mouse strains with functional, nonfunctional, or degenerated rods and cones show that DENAQ is effective only in retinas with degenerated photoreceptors. DENAQ confers light sensitivity on a hyperpolarization-activated inward current that is enhanced in degenerated retina, enabling optical control of retinal ganglion cell firing. The acceptable light sensitivity, favorable spectral sensitivity, and selective targeting to diseased tissue make DENAQ a prime drug candidate for vision restoration in patients with end-stage RP and AMD.


Figure 1
Figure 1. The red-shifted photoswitch DENAQ restores light responses to blind retinas from rd1 mice
(A) Molecular structure of DENAQ. Light converts DENAQ from the trans to the cis form, and the compound quickly relaxes back to the trans form in the dark. (B–C) MEA recording from an rd1 retina before DENAQ treatment (B) and after treatment with 300 µM DENAQ (C). Light (white) and dark (black/gray) episodes are shown. (D) Spectral sensitivity of the DENAQ-mediated light response. (E) LRI value distributions for RGCs from untreated (black) (median LRI=0, n=12 retinas) and DENAQ-treated (blue) rd1 retinas (median LRI=0.42, n=12 retinas, p<0.001, rank sum test). (F–G) DENAQ restored light responses in every retina tested. Mean RGC firing rate for each retina in light and darkness before (n=12 retinas, p=0.94) (F) and after DENAQ treatment (n=12 retinas, p<0.001) (G). Mean±SEM values are shown in red.
Figure 2
Figure 2. Intensity requirement for restoring light responses and persistence of DENAQ in the eye
(A) Light intensity vs. response curve for DENAQ (n=5 retinas). Data are mean±SEM. Labeled dotted lines represent the thresholds for activation of DENAQ (blue), ChR2 (Thyagarajan et al., 2010) (red), NpHR (Busskamp et al., 2010) (red) and AAQ (Polosukhina et al., 2012) (red) in the retina. (B) MEA recording from a DENAQ-treated rd1 retina stimulated with moderate intensity (3×1014 photons/cm2/sec) white light. Light (white) and dark (black/gray) episodes are shown. (C) Persistence of photosensitization elicited by in vivo injection of AAQ (red, half-life=3.6 hours) and DENAQ (blue, half-life=2.1 days). Responses were measured on the MEA ex vivo, hours to days after in vivo injection of 2 µL of 20 mM photoswitch (n=4 retinas per time point). Photoswitch half-life was calculated by fitting the data with a monoexponential decay function.
Figure 3
Figure 3. The DENAQ-treated rd1 retina generates spatially precise light responses
(A) Targeted illumination of a portion of an rd1 retina centered on a single MEA electrode (top). Electrode E4 was stimulated with a 60 µm diameter light spot. Only the targeted electrode recorded a large increase in RGC firing in response to white light (bottom). PI values are color-coded (scale at left) and also represented by bar height. The orange bar is electrode E4. Empty squares are electrodes on which no action potentials were recorded. (B) Targeted illumination elicits an increase in activity in stimulated RGCs and has no effect on surrounding RGCs (n = 16 cells and n = 741 cells, respectively, from seven retinas). LRI values of RGCs (black circles) as a function of distance from the target electrode, displayed in 200 µm bins. Median plus and minus the 95% confidence intervals are shown in red. (C) Responses of DENAQ-treated rd1 RGCs to stimulation with spots of light of increasing diameter. The light response saturates at 240 µm diameter spot size. Data are mean±SEM, n=20 cells. See also Table S1.
Figure 4
Figure 4. DENAQ only photosensitizes retinas in which rod and cone photoreceptors have degenerated
(A–B) MEA recording from a WT retina before (A) and after (B) treatment with 300 µM DENAQ. Light (white) and dark (black/gray) episodes are shown. (C) RGC PLRI values for WT retinas before (black) and after (blue) DENAQ treatment (n=6 retinas, p=0.34, rank sum test). (D) Light responses of untreated and DENAQ-treated WT (n=6, p=0.72), TKO (n=6, p=0.97), rd1 (n=12, p<0.001) and rd4 retinas (n=6, p<0.001). Mean PLRI values are shown for WT retinas and mean LRI values for TKO, rd1 and rd4 retinas. Data are mean±SEM.
Figure 5
Figure 5. DENAQ selectively photosensitizes RGCs from retinas with degenerated photoreceptors
(A–B) MEA recording from pharmacologically isolated rd1 (A) and WT (B) RGCs. (C) Light responses of pharmacologically isolated WT RGCs (mean LRI=−0.01, n=10 retinas, p<0.001), TKO RGCs (mean LRI=0, n=6 retinas, p<0.001), rd1 RGCs (mean LRI=0.70, n=8 retinas) and rd4 RGCs (mean LRI=0.43, n=6 retinas). See also Figures S1 and S2.
Figure 6
Figure 6. DENAQ selectively photosensitizes Ih in rd1 but not WT RGCs
(A–B) Current vs. voltage relationships of RGCs from WT (A) and rd1 (B) retinas obtained in darkness (black) or in light (green or blue). (C) Light-elicited current, defined as the difference between currents measured in light and dark in the WT RGC (from panel A) (green) and in the rd1 RGC (from panel B) (blue). (D) Light-elicited change in Ih (left), and in IK (right) in WT RGCs (black) (n=11) and in rd1 RGCs (blue) (n=9), shown as percent difference. Ih was photoregulated in rd1 (mean=22%) RGCs, but not in WT RGCs (mean=−0.3%, p<0.001). IK was photosensitized to the same extent in WT and rd1 RGCs (WT mean=15%, rd1 mean=23%, p=0.2). (E) Ih and IK current densities in WT (black) (n=13 cells) and rd1 (blue) (n=7) RGCs. Ih density was significantly higher in rd1 RGCs (mean=6.4 pA/pF) than in WT RGCs (mean=2.9 pA/pF, p<0.01). IK density was not significantly different (WT mean=12.3 pA/pF, rd1 mean=16.6 pA/pF, p=0.3). (F) DENAQ treatment photosensitizes rd1 RGCs (n=8 retinas, mean firing rate increase (MFRI)=3.5×, p<0.001) (left). Subsequent application of Ih blockers - ZD7288 (n=7 retinas, MFRI=1.18, p=0.38) (second from left), ivabradine (n=4 retinas, MFRI=1.01, p=0.87) (second from right) or cilobradine (n=4 retinas, MFRI=1.02, p=0.81) (right) abolishes DENAQ-mediated rd1 RGC photosensitization. Mean±SEM firing rates are shown in red. (G) Mean light response index of DENAQ-treated rd1 RGCs (mean LRI=0.7) (blue). Ih blockers - ZD7288 (mean LRI=0.02, p<0.001) (black), ivabradine (mean LRI=0, p<0.001) (red) and cilobradine (mean LRI=0.01, p<0.001) (green) eliminate DENAQ-mediated RGC photosensitization. Data are mean±SEM. See also Figures S3 and S4.
Figure 7
Figure 7. DENAQ restores light-modulated open field locomotor behavior in blind mice
(A) Movement trajectory of the same rd1 mouse exploring an open cylindrical cage in the dark (gray) and under 500 nm light (green) before (top) and after (bottom) DENAQ injection. (B) Cumulative distance traveled by an rd1 mouse before (left) and after (right) DENAQ injection. (C) Bar graph of activity in the light divided by activity in darkness before (black) (n=20 mice, mean=1.04) and after intravitreal injection of DENAQ and before (yellow) or after (gray) sham injection (n=6 mice, p=0.88). Activity ratios for the first 100 sec of exploratory behavior of DENAQ-injected mice (blue) (mean=1.73, p<0.001), 100–200 sec in the light (green) (mean=1.52, p<0.001) and the final 100 sec in the light (red) (200–300 sec) (mean=1.39, p<0.05). The amount of exploratory activity decreased with time. Data are mean±SEM. See also Figure S5.
Figure 8
Figure 8. DENAQ enables visual learning in blind mice
(A) Diagram of the paired, unpaired conditioning and recall protocols for the visual fear conditioning assay. Electric foot shock (yellow) and light flash (blue) episodes are shown. (B) Cumulative distance traveled by a paired-conditioned sham-injected rd1 mouse (left, black), a paired-conditioned DENAQ-injected rd1 mouse (middle, blue) and a paired-conditioned WT mouse (right, red) in the 30 sec of darkness preceding the light flash and the subsequent 30 sec in the light during the recall trial. (C) Bar graph of activity in light divided by activity in darkness for sham-injected rd1 (n=9, p=0.71), DENAQ-injected rd1 (n=10, p<0.001) and WT mice (n=10, p<0.001) during the recall trial. Data are mean±SEM.

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