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.

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.

High frequency neural spiking and auditory signaling by ultrafast red-shifted optogenetics.

Optogenetics revolutionizes basic research in neuroscience and cell biology and bears potential for medical applications. We develop mutants leading to a unifying concept for the construction of various channelrhodopsins with fast closing kinetics. Due to different absorption maxima these channelrhodopsins allow fast neural photoactivation over the whole range of the visible spectrum. We focus our functional analysis on the fast-switching, red light-activated Chrimson variants, because red light has lower light scattering and marginal phototoxicity in tissues. We show paradigmatically for neurons of the cerebral cortex and the auditory nerve that the fast Chrimson mutants enable neural stimulation with firing frequencies of several hundred Hz. They drive spiking at high rates and temporal fidelity with low thresholds for stimulus intensity and duration. Optical cochlear implants restore auditory nerve activity in deaf mice. This demonstrates that the mutants facilitate neuroscience research and future medical applications such as hearing restoration.

The synaptic ribbon is critical for sound encoding at high rates and with temporal precision.

We studied the role of the synaptic ribbon for sound encoding at the synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) in mice lacking RIBEYE (RBEKO/KO). Electron and immunofluorescence microscopy revealed a lack of synaptic ribbons and an assembly of several small active zones (AZs) at each synaptic contact. Spontaneous and sound-evoked firing rates of SGNs and their compound action potential were reduced, indicating impaired transmission at ribbonless IHC-SGN synapses. The temporal precision of sound encoding was impaired and the recovery of SGN-firing from adaptation indicated slowed synaptic vesicle (SV) replenishment. Activation of Ca2+-channels was shifted to more depolarized potentials and exocytosis was reduced for weak depolarizations. Presynaptic Ca2+-signals showed a broader spread, compatible with the altered Ca2+-channel clustering observed by super-resolution immunofluorescence microscopy. We postulate that RIBEYE disruption is partially compensated by multi-AZ organization. The remaining synaptic deficit indicates ribbon function in SV-replenishment and Ca2+-channel regulation.