Channelrhodopsin variants for high-rate optogenetic neurostimulation at low light intensities

Optogenetics allows versatile control of excitable cell networks, which advances basic science research and drives the development of future medical applications. Fast-closing channelrhodopsins (ChRs) are required for high temporal fidelity of neurostimulation, but their short channel open times require sufficient plasma membrane expression and high light intensity, challenging clinical translation. Here, we addressed the need of high-rate neurostimulation by engineering optimized blue-light-sensitive ChR variants. In particular, we report on the ChR2 variant f-ChR2 TC enabling high frequency stimulation at low light requirements, due to its good plasma membrane targeted expression and balanced closing kinetics. Upon Adeno-associated virus (AAV) mediated f-ChR2 TC expression in spiral ganglion neurons of the inner ear in mice, f-ChR2 TC accordingly enabled optogenetic stimulation of the auditory nerve with sizeable responses beyond 300 Hz and low pulse energy thresholds. Translating the approach to the larger cochlea of gerbils, we tested the utility of f-ChR2 TC for evaluating multichannel optical cochlear implants with blue light emitting diodes and found light-efficient stimulation of the auditory pathway by single LEDs at rates ≥100 Hz.