Investigating activity-dependent synaptic plasticity at cochlear inner hair cells in ribbon synapses

Hearing in mammals relies on the ultrafast, precise, and specific encoding of sound into electrochemical signals for the brain, a function critically mediated by inner hair cell (IHC) ribbon synapses. Structural plasticity of these synapses may play a significant role in optimizing auditory signal transmission, however the underlying mechanisms remain unclear. This thesis aimed to establish and refine experimental methodologies for investigating activity-dependent structural plasticity at IHC ribbon synapses during auditory system development, a period marked by spontaneous activity fundamental for neuronal survival, circuit formation, and maturation. Organotypic cultures of the organ of Corti were prepared from wild-type (WT) and Ai32-VC-KI optogenetic mouse lines, allowing for long-term experimentation. Spontaneous activity in these cultures was characterized via calcium imaging, revealing persistent, low-frequency activity comparable to in vivo observations, suggesting small interference with stimulation studies. Pharmacological inhibition of P2YR1-mediated spontaneous activity using MRS2500 successfully silenced these calcium waves without cytotoxic effects, confirming their origin in supporting cells. Additionally, the performance of a custom-built optogenetic stimulation device was evaluated, showing limitations in temperature stability and power output, but no acute toxic effects on cultured tissue. Immunohistochemistry for the immediate early gene cFos indicated its suitability as a marker for successful activation upon chemical stimulation and could be useful in future optogenetic experiments. Overall, this work establishes a reliable in vitro model and provides methodological groundwork for future studies probing activity-dependent synaptic plasticity at IHC ribbon synapses.