Characterizing a Novel Powerful Channelrhodopsin Variant for Preclinical Studies on Optogenetic Cochlear Implants
by Fadhel El May
Date of Examination:2024-12-18
Date of issue:2025-10-29
Advisor:Prof. Dr. Tobias Moser
Referee:Prof. Dr. Tobias Brügmann
Referee:Prof. Dr. Stefan Treue
Persistent Address:
http://dx.doi.org/10.53846/goediss-11494
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Abstract
English
Contemporary electrical cochlear implants (eCI) directly stimulate spiral ganglion neurons (SGNs) using electric current to partially restore auditory function in patients diagnosed with profound sensorineural hearing loss or deafness. The majority of the >1 Mio eCI users are able to understand speech in quiet environments. However, improved speech understanding in daily acoustic situations with heightened background noise remains an unmet medical need. This shortcoming is attributed to the limited number of independent stimulation channels due to the broad spread of current from each electrode. As light can be conveniently confined in space, optogenetic stimulation of SGNs has been proposed for increasing the number of independent channels. Multiunit recordings from the inferior colliculus (IC) of anaesthetized animals demonstrated better spectral selectivity of optogenetic CIs (oCIs) compared to eCI. Yet, the energy threshold was higher for oCI compared to eCI challenging the energy budget. To address this shortcoming of optogenetic stimulation, we engineered and characterized a novel and powerful variant of the channelrhodopsin (ChR) ChRmine, that we called ChReef to increase the light sensitivity of SGNs. I investigated the properties of ChReef-mediated stimulation of the auditory pathway by recordings of midbrain multiunit activity in adult Mongolian gerbils which were subjected to cochlear injections of adeno-associated virus (AAV) at the age of postnatal days 6-8 to express ChReef in SGNs. In a first step, I used an optical fiber to stimulate the cochlea with green or blue light. I found that IC activity in the context of ChReef-mediated optogenetic stimulation of the cochlea using green light stimulation had the lowest radiant flux threshold when compared to other ChRs. Consequently, ChReef enabled higher maximal neural response strength at low radiant fluxes than what was reported previously using different ChRs while the dynamic range (DR) was comparable. To disentangle SGN stimulation from potential co-stimulation of hair cells, I acutely deafened animals using ototoxic agent kanamycin. I found that optogenetic stimulation restored IC activity yet with nonsignificant increase in IC thresholds and decrease in maximal neural response strength and DR, possibly attributable to the deafening methodology. In a second step, I used IC recordings from gerbils expressing ChReef in SGNs to characterize optogenetic stimulation by microfabricated oCI. I employed either oCIs based on 60x60 µm2 blue µLED arrays or on 220×270 µm2 commercial green LEDs (CreeLEDs) bonded on flexible polyimide carriers. We established a reliable quality control setup combining power calibration with camera LED analysis and discovered technical challenges of blue µLED oCIs. Nonetheless, I could use them to demonstrate ChReef-mediated SGN stimulation by µLEDs. I then moved to CreeLED-based oCIs and found that green CreeLEDs elicited robust IC responses and allowed cochlear stimulation at different tonotopic places. IC thresholds were not significantly different to those of optical fiber stimulation. We did not detect distinct spatial tuning curves upon dual LED stimulation but stimulation by a block of LED indicated increase of the spread of excitation. Acute deafening by kanamycin did not impact thresholds significantly. The results of my thesis show the potential of ChReef as a promising ChR candidate for further developments toward future clinical optogenetic hearing restoration.
Keywords: auditory neuroscience; optogenetics; cochlear implant; neuroprosthetics; sensory neuroscience; optical stimulation; gene therapy; hearing restoration
