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AAV-mediated optogenetic neural stimulation has become a clinical approach for restoring function in sensory disorders and feasibility for hearing restoration has been indicated in rodents. Nonetheless, long-term stability and safety of AAV-mediated channelrhodopsin (ChR) expression in spiral ganglion neurons (SGNs) remained to be addressed. Here, we used longitudinal studies on mice subjected to early postnatal administration of AAV2/6 carrying fast gating ChR f-Chrimson under the control of the human synapsin promoter unilaterally to the cochlea. f-Chrimson expression in SGNs in both ears and the brain was probed in animals aged 1 mo to 2 yr. f-Chrimson was observed in SGNs at all ages indicating longevity of ChR-expression. SGN numbers in the AAV-injected cochleae declined with age faster than in controls. Investigations were extended to the brain in which viral transduction was observed across the organ at varying degrees irrespective of age without observing viral spread-related pathologies. No viral DNA or virus-related histopathological findings in visceral organs were encountered. In summary, our study demonstrates life-long (24 mo in mice) expression of f-Chrimson in SGNs upon single AAV-dosing of the cochlea., (© 2022 Bali et al.)

In the last 15 years, optogenetics has revolutionized the life sciences and enabled studies of complex biological systems such as the brain. Applying optogenetics also has great potential for restorative medicine, such as hearing restoration, by stimulating genetically modified spiral ganglion neurons of the cochlea with light. To this end, opsins with short closing kinetics are required, given the high firing rates and utmost temporal precision of spiking in these neurons. Chronos is the fastest native blue channelrhodopsin (ChR) reported so far with a closing kinetics bellow 1 ms at body temperature and an interesting candidate for the development of the future optogenetic cochlear implants. This book chapter explains in more details the development and application of Chronos with optimized membrane targeting for temporally precise optical stimulation of spiral ganglion neurons. In addition, the generation of adeno-associated virus (AAV) and AAV delivery to the cochlea of postnatal mice and the procedure to record optically evoked auditory brainstem responses are described.

Optogenetic tools, providing non-invasive control over selected cells, have the potential to revolutionize sensory prostheses for humans. Optogenetic stimulation of spiral ganglion neurons (SGNs) in the ear provides a future alternative to electrical stimulation used in cochlear implants. However, most channelrhodopsins do not support the high temporal fidelity pertinent to auditory coding because they require milliseconds to close after light-off. Here, we biophysically characterized the fast channelrhodopsin Chronos and revealed a deactivation time constant of less than a millisecond at body temperature. In order to enhance neural expression, we improved its trafficking to the plasma membrane (Chronos-ES/TS). Following efficient transduction of SGNs using early postnatal injection of the adeno-associated virus AAV-PHPB into the mouse cochlea, fiber-based optical stimulation elicited optical auditory brainstem responses (oABR) with minimal latencies of 1 ms, thresholds of 5 μJ and 100 μs per pulse, and sizable amplitudes even at 1,000 Hz of stimulation. Recordings from single SGNs demonstrated good temporal precision of light-evoked spiking. In conclusion, efficient virus-mediated expression of targeting-optimized Chronos-ES/TS achieves ultrafast optogenetic control of neurons., (© 2018 The Authors.)