Ultrafast optogenetic stimulation of the auditory pathway by targeting-optimized Chronos.

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.

Graded optogenetic activation of the auditory pathway for hearing restoration.

Optogenetic control of neural activity enables innovative approaches to improve functional restoration of diseased sensory and motor systems. For clinical translation to succeed, optogenetic stimulation needs to closely match the coding properties of the targeted neuronal population and employ optimally operating emitters. This requires the customization of channelrhodopsins, emitters and coding strategies. Here, we provide a framework to parametrize optogenetic neural control and apply it to the auditory pathway that requires high temporal fidelity of stimulation. We used a viral gene transfer of ultrafast targeting-optimized Chronos into spiral ganglion neurons (SGNs) of the cochlea of mice. We characterized the light-evoked response by in vivo recordings from individual SGNs and neurons of the anteroventral cochlear nucleus (AVCN) that detect coincident SGN inputs. Our recordings from single SGNs demonstrated that their spike probability can be graded by adjusting the duration of light pulses at constant intensity, which optimally serves efficient laser diode operation. We identified an effective pulse width of 1.6 ms to maximize encoding in SGNs at the maximal light intensity employed here (∼35 mW). Alternatively, SGNs were activated at lower energy thresholds using short light pulses (<1 ms). An upper boundary of optical stimulation rates was identified at 316 Hz, inducing a robust spike rate adaptation that required a few tens of milliseconds to recover. We developed a semi-stochastic stimulation paradigm to rapidly (within minutes) estimate the input/output function from light to SGN firing and approximate the time constant of neuronal integration in the AVCN. By that, our data pave the way to design the sound coding strategies of future optical cochlear implants.

Application of Targeting-Optimized Chronos for Stimulation of the Auditory Pathway.

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.

Developing Fast, Red-Light Optogenetic Stimulation of Spiral Ganglion Neurons for Future Optical Cochlear Implants.

Optogenetic stimulation of type I spiral ganglion neurons (SGNs) promises an alternative to the electrical stimulation by current cochlear implants (CIs) for improved hearing restoration by future optical CIs (oCIs). Most of the efforts in using optogenetic stimulation in the cochlea so far used early postnatal injection of viral vectors carrying blue-light activated channelrhodopsins (ChRs) into the cochlea of mice. However, preparing clinical translation of the oCI requires (i) reliable and safe transduction of mature SGNs of further species and (ii) use of long-wavelength light to avoid phototoxicity. Here, we employed a fast variant of the red-light activated channelrhodopsin Chrimson (f-Chrimson) and different AAV variants to implement optogenetic SGN stimulation in Mongolian gerbils. We compared early postnatal (p8) and adult (>8 weeks) AAV administration, employing different protocols for injection of AAV-PHP.B and AAV2/6 into the adult cochlea. Success of the optogenetic manipulation was analyzed by optically evoked auditory brainstem response (oABR) and immunohistochemistry of mid-modiolar cryosections of the cochlea. In order to most efficiently evaluate the immunohistochemical results a semi-automatic procedure to identify transduced cells in confocal images was developed. Our results indicate that the rate of SGN transduction is significantly lower for AAV administration into the adult cochlea compared to early postnatal injection. SGN transduction upon AAV administration into the adult cochlea was largely independent of the chosen viral vector and injection approach. The higher the rate of SGN transduction, the lower were oABR thresholds and the larger were oABR amplitudes. Our results highlight the need to optimize viral vectors and virus administration for efficient optogenetic manipulation of SGNs in the adult cochlea for successful clinical translation of SGN-targeting gene therapy and of the oCI.

Utility of red-light ultrafast optogenetic stimulation of the auditory pathway.

Optogenetic stimulation of spiral ganglion neurons (SGNs) in the ear provides a future alternative to electrical stimulation used in current cochlear implants. Here, we employed fast and very fast variants of the red-light-activated channelrhodopsin (ChR) Chrimson (f-Chrimson and vf-Chrimson) to study their utility for optogenetic stimulation of SGNs in mice. The light requirements were higher for vf-Chrimson than for f-Chrimson, even when optimizing membrane expression of vf-Chrimson by adding potassium channel trafficking sequences. Optogenetic time and intensity coding by single putative SGNs were compared with coding of acoustic clicks. vf-Chrimson enabled putative SGNs to fire at near-physiological rates with good temporal precision up to 250 Hz of stimulation. The dynamic range of SGN spike rate coding upon optogenetic stimulation was narrower than for acoustic clicks but larger than reported for electrical stimulation. The dynamic range of spike timing, on the other hand, was more comparable for optogenetic and acoustic stimulation. In conclusion, f-Chrimson and vf-Chrimson are promising candidates for optogenetic stimulation of SGNs in auditory research and future cochlear implants.