In vitro and in vivo characterization of improved channelrhodopsin ChRmine variants for optogenetic activation of the auditory pathway

Background Optogenetic activation of the auditory pathway using channelrhodopsins (ChR) presents a promising solution to overcome the limitations of cochlear implants imposed by the wide-spread electrical excitation of spiral ganglion neurons (SGNs). Before a clinical translation of optogenetic hearing restoration a suitable ChR variant needs to be identified. In this study, we designed and investigated improved variants of the green-light activated ChRmine for their optogenetic utility.

Methods In vitro characterization of electrophysiological properties of ChRmine mutants was performed by whole cell patch-clamp recordings of transfected neuroma glioblastoma cells expressing one of 3 mutants or wild-type (WT). For in vivo characterization, adeno-associated-viruses carrying ChRmine mutant#3 (n=11) or WT (n=8) were injected into the round window of neonatal C57Bl6/J mice. 6-10 weeks after injection optically evoked auditory brainstem responses (oABRs) were measured.

Results Patch-clamp recordings of all variants showed a decrease in desensitization compared to the WT as well as large stationary photocurrent densities and high light sensitivity, but comparatively slow closing kinetics. oABRs were elicited in all animals with amplitudes ranging 8-15 μV and mean thresholds of 0.79±0.4 mW (mutant#3) and 5.7±6.4 mW (WT) upon 1 ms stimulation at 10 or 20 Hz with light at 594 nm.

Conclusion ChRmine mutant#3 exhibits large photocurrents promising robust neuronal photoactivation at moderate ChR expression levels and low light intensities. These are favorable ChR properties for clinical application of optogenetic hearing restoration as they lower the power requirements of a future optical cochlear implant. Further work needs to focus on the acceleration of the ChR closing kinetics.

Publication History

Article published online:
12 May 2023