Photopharmacologic Vision Restoration Reduces Pathological Rhythmic Field Potentials in Blind Mouse Retina

Photopharmacology has yielded compounds that have potential to restore impaired visual responses resulting from outer retinal degeneration diseases such as retinitis pigmentosa. Here we evaluate two photoswitchable azobenzene ion channel blockers, DAQ and DAA for vision restoration. DAQ exerts its effect primarily on RGCs, whereas DAA induces light-dependent spiking primarily through amacrine cell activation. Degeneration-induced local field potentials remain a major challenge common to all vision restoration approaches. These 5–10 Hz rhythmic potentials increase the background firing rate of retinal ganglion cells (RGCs) and overlay the stimulated response, thereby reducing signal-to-noise ratio. Along with the bipolar cell-selective photoswitch DAD and second-generation RGC-targeting photoswitch PhENAQ, we investigated the effects of DAA and DAQ on rhythmic local field potentials (LFPs) occurring in the degenerating retina. We found that photoswitches targeting neurons upstream of RGCs, DAA (amacrine cells) and DAD (bipolar cells) suppress the frequency of LFPs, while DAQ and PhENAQ (RGCs) had negligible effects on frequency or spectral power of LFPs. Taken together, these results demonstrate remarkable diversity of cell-type specificity of photoswitchable channel blockers in the retina and suggest that specific compounds may counter rhythmic LFPs to produce superior signal-to-noise characteristics in vision restoration.

Recordings were made under control conditions (black), following the application of K + -channel antagonists (red), and after washing out the drugs (blue). Spike rate was strongly attenuated during the light flash following drug application. (B) The photoswitch index was significantly suppressed following application of K + -channel antagonists (n = 9 cells; p = 2.0 x 10 -2 ). (C) Excitatory and inhibitory synaptic current recordings in the same cell following application of DAQ. Synaptic currents were relatively small. (D) On average, the ionotropic glutamate receptor antagonists suppressed ganglion cell spike responses by ~25%, but this effect was not statistically significant (n = 5 cells; p = 0.16). (E) The photoswitch index was not significantly decreased following application of ionotropic glutamate receptor antagonists that attenuated excitatory synaptic input to the recorded ganglion cells (n = 5 cells; p = 0.25). Bars indicate mean ± SEM. All statistical tests were paired and were performed using the Wilcoxon signed rank test. Control conditions refer to conditions where only the photoswitch DAQ is applied.

Chemical Synthesis General Experimental Techniques
Unless stated otherwise all reactions were carried out with magnetic stirring using oven-dried glassware (160 °C) under inert gas atmosphere (nitrogen or argon). Syringes used to transfer reagents and solvents were purged with nitrogen prior to use. Low temperature reactions were carried out in a Dewar vessel filled with the appropriate cooling agent e.g. H2O/ice (0 °C). Heating was conducted using a heated oil bath. Yields refer to spectroscopically pure compounds unless otherwise stated.

Solvents and Reagents
Reaction solvents were purchased from Acros Organics as 'extra dry' over molecular sieves and handled under inert gas atmosphere. Tetrahydrofuran (THF) was distilled from Na/benzophenone prior to use. Dichloromethane (DCM), triethylamine (TEA) and diisopropylethylamine (DIPEA) were distilled from calcium hydride. Ethanol was purchased from commercial suppliers and used as received. Solvents for extraction and flash column chromatography were purchased in technical grade purity and distilled under reduced pressure on a rotary evaporator prior to use. All other reagents and solvents were purchased from commercial suppliers and used as received.

Chromatography
Reactions and chromatography fractions were monitored by qualitative thin-layer chromatography (TLC) on silica gel F254 TLC plates from Merck KGaA. Analytes were visualized by irradiation with UV light and/or by immersion of the TLC plate in ninhydrin or potassium permanganate solution followed by heating with a hot-air gun. Flash column chromatography was performed Geduran® Si60 (40-63 µm) silica gel from Merk KGaA (eluents are given in parenthesis). Reverse Phase column chromatography was performed on Waters C18 (C18; 55-105 µm, 125 Å) as stationary phase (eluents are given in parenthesis). LC-MS was performed on an Agilent 1260 Infinity HPLC System, MS-Agilent 1100 Series, Type: 1946D, Model: SL, equipped with a Agilent Zorbax Eclipse Plus C18 (100 x 4.6 mm, particle size 3.5 micron) reverse phase column with a constant flow-rate of 1 mL/min and a 10 → 100% MeCN/H2O + 0.1% FA gradient over 10 min.

NMR Spectra
NMR spectra were measured on Varian 400 MHz Bruker AVIII HD (cryoprobe) for proton nuclei (100 MHz for carbon nuclei respectively). The 1 H NMR shifts are reported in parts per million (ppm) related to the chemical shift of tetramethylsilane. 1 H and 13 C NMR shifts were calibrated to the residual solvent signals: CDCl3 (7.26 ppm/77.16 ppm) and DMSO (2.50 ppm/39.5 ppm). 1 H NMR spectroscopic data are reported as follows: Chemical shift in ppm (multiplicity, coupling constants (Hz), integration). The multiplicities are abbreviated as follows: s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet) and are reported as observed. Except for multiplets, the chemical shift of all signals is reported as the centre of the resonance range. Additionally, to 1 H and 13 C NMR measurements, 2D NMR techniques as homonuclear correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond S12 coherence (HMBC) were used to assist signal assignment. All raw fid files were processed, and the spectra analyzed using the program MestReNova from Mestrelab Research S. L.

Mass Spectra
All high-resolution mass spectra (HRMS) were recorded by the LMU Mass Spectrometry Service. HRMS were recorded on a MAT 90 from Thermo Finnigan GmbH using electrospray ionisation (ESI) or a MAT 90 from Jeol Ltd. using electron ionization (EI).

UV/Vis Spectra
UV/Vis spectra were recorded on a Varian Cary 50 Scan UV/Vis spectrometer using Helma SUPRASIL precision cuvettes (10 mm light path). All compound stock solutions were prepared under benchtop light conditions at 50 mM in DMSO and diluted to the right concentration in the final solvent (DMSO or PBS). Photoswitching was achieved using a Polychrome V (Till Photonics) Monochromator, or a Prizmatix ultra high power LED (460 nm), connected to a fiberoptic cable through which the sample in the spectrophotometer was irradiated from the top.