Establishment of a test system for an optogenetic biohybrid cochlear implant

The cochlear implant is the world's most successful neuroprosthetic device. The electrode directly stimulates the spiral ganglion neurons (SGN), the first neurons in the auditory system, in the cochlea at different places, which corresponds to a different characteristic frequency. A limiting performance factor of an electric cochlear implant (eCI) is the crosstalk of channel-interaction due to spread of excitation, resulting in poor speech perception in background noise and difficulty appreciating complex sounds such as music. An optical cochlear implant (oCI) may overcome these limitations, as light can be focused much more precise to activate a narrow band of SGNs. This thesis presents a novel way of stimulating SGNs via a neuronal-tandem trigger approach, which was developed during this thesis. Inter-neuronal communication between spiral ganglion neurons and neurons of other functional units, in this case cortex neurons (CN) and hippocampus neurons (HN) is probed via an all-optical system, consisting of a blue-shifted channelrhodopsin variant (CheRiff) with a genetically encoded calcium indicator, namely jRCaMP1a, which has its emission spectra in the red light range. This enabled for simultaneous optical stimulation and recording from spatially separated small neuronal populations. A manipulation LED was fixed directly above either a CheRiff transduced HN or a CN population. Three spots per population were stimulated with four different pulse durations (10 ms; 100 ms; 250 ms; 500 ms). Each recording consisted of 10 pulses of blue light separated by an interval of 5.5 s. Simultaneously, calcium imaging with continuous green light illumination (570 nm, 15 mW/cm2) was performed. The stimulation of SGNs via a neuronal-tandem approach was possible for the first time with both optogenetic manipulated HNs and CNs respectively. SGNs (that are not transduced with CheRiff) are activated when nearby populations of cortical or hippocampal cultured neurons are stimulated with light, indicating that the neurons of different origin are inter-connected and communication takes place between them. Another observation made was, that the stimulation efficiency of the SGNs generally increased with the stimulation length and reached a plateau at 250 ms, whereas longer stimulation pulses did not result in stronger calcium signals. This fact could indicate an effect called excitotoxicity. Further, a structured cell growth system based on gold-coated coverslips (AuCS) and poly(ethylene glycol) methyl ether thiol (mPEG) was established, to enable spatially directed signal generation. The mPEG generate a cell repellent surface on the AuCS and laser irradiation generates the specified direction of growth. First optogenetic stimulation experiments with HL-1 ChR2 cells showed propagating calcium waves after LED irradiation within the designated structure, pointing out the great reliability of this method. Together, the neuronal-tandem approach developed during this thesis could pave the way to a completely new type of an oCI