Azobenzene-based DENAQ photoswitch confers light sensitivity to degenerated but not to healthy canine retina

Abstract

Purpose : To determine if light responses in degenerated canine retinas can be restored by ex vivo application of DENAQ, a photoisomerizable compound that has recently been shown to confer light sensitivity to voltage-gated ion channels in retinal ganglion cells of the degenerated murine retina.

Methods : A standard multi (60)-electrode array system was used to record retinal ganglion cell (RGC) firing activity in retinal explants from 5 degenerated [mutant: 4 rcd1/PDE6B (age: 2.1-7.7 yrs), 1 xlpra2/RPGR (age: 1.1 yr)] and 6 WT (age: 0.3-6.6 yrs) retinas treated with DENAQ (1 mM). Synaptic blockers were used to isolate retinal ganglion cell responses after DENAQ application. Light responses were evoked with calibrated flashes of 455 nm light. Spike sorting was performed in Plexon^TM and results were further analyzed using custom programs developed in Matlab^TM.

Results : All degenerated retinas (except 1 rcd1) did not show light-dependent spiking activity before in vitro DENAQ application. Following DENAQ treatment, retinas demonstrated strong ON-responses to 455 nm blue light. Application of synaptic blockers further enhanced DENAQ-driven responses by suppressing background firing. Responses from the DENAQ-treated 7.7 yr-old rcd1 retina and from the xlpra2 retina had significantly reduced sensitivity. All other cases had responses to light at intensities as low as 0.2 mW/cm² using 1s or 5s flashes (0.1 Hz), and response amplitude saturated above 12 mW/cm². Recovery of DENAQ driven responses had a time constant of ~ 3 s. Up to 30% of cells responsive to a slow stimulation after DENAQ application (1 or 5 s flashes at 0.1 Hz) also responded to a 4 Hz flicker stimulation with a threshold around 3 mW/cm². No responses where observed in DENAQ treated WT retinas at dim and moderate intensities (up to 6 mW/cm²).

Conclusions : Similar to previously reported findings in mice, DENAQ treatment conferred light-sensitivity to RGCs in the degenerated but not WT canine retina. Light responses were observed at intensity levels which can be encountered under natural conditions (as low as 0.2 mW/cm²). Treated retinas were responsive to 4 Hz flicker stimulations. Flicker sensitivity was around 10x lower compared to the sensitivity to discrete flashes. Efficiency of DENAQ treatment may depend on the animal’s age/stage of inner retinal degeneration, and genotype.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.