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K. P. Greenberg, A. Pham, F. S. Werblin; A Side-By-Side Comparison of the Next Generation Optical Neuromodulators of Retinal Activity. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3467. doi: https://doi.org/.
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The insertion of optical neuromodulators into normally non-photosensitive retinal neurons is a promising approach for restoring sight to profoundly blind individuals. However, improvements in depolarizing (i.e. channelrhodopsin) and hyperpolarizing (i.e. halorhodopsin) opsin performance are necessary before this therapeutic approach becomes clinically viable. We evaluated threshold sensitivity, peak/plateau responses, dynamic range, temporal responses, expression levels, and membrane trafficking: parameters essential for the selection of neuromodulators well suited to visual prosthesis.
Coding regions corresponding to hChR2, step function opsins hChR2(C128A/S/T), oChEF, oChIEF, VChR1, LiGLuR, LNMDAR, Opto-A1AR, Opto-B2AR, eNpHR, MAC, Arch, and Hylighter were cloned into identical vector constructs containing the Camk2α promoter and WPRE. Constructs were delivered to ganglion cells in organotypic rabbit retina explants by biolistic (gene-gun) mediated transfection. Twenty-four hours following gene transfer, light mediated spiking and currents were measured in intact retinae via extracellular and whole-cell patch clamp recording. Trafficking of opsins to the membrane and presence of ER aggregations or toxicity was evaluated by confocal imaging.
The minimum threshold for sustained oChIEF spiking required 0.2mW/mm2, approximately 10 fold less irradiance than required by hChR2. Plateau current responses from oChIEF and Arch were 4-5 fold larger than hChR2 and eNpHR. The dynamic range of Arch was nearly 3 logs, while eNpHR responses saturated at 2 logs. LiGluR, oChEF, and oChIEF showed superior 50Hz pulse response kinetics to hChR2. Opto-A1AR and Opto-B2AR both generated excitatory photocurrents at negative holding potentials. The nAChR N-terminal and Kir2.1 C-terminal ER export signal peptides made significant improvements in eNPHR membrane trafficking, though these failed to improve Arch. However, the prolactin signal sequence and MHC Class I antigen enabled substantial improvements in Arch membrane trafficking. Expression levels of these opsins were comparable, with improved membrane localization resulting in decreased toxicity and increased photocurrents.
The next generation of optical neuromodulators demonstrates dramatic improvements in threshold sensitivity, dynamic range, plateau responses, kinetics, and membrane trafficking. These improvements bring us ever closer to achieving a practical optical neuromodulator-based prosthetic for profound vision loss.
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