Lina Hofmann

• Inhibitory transmission in the auditory brainstem is essential for sound source localization. Glycinergic synapses between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO) process interaural level differences and reliably transmit inhibitory signals, even during minutes of high-frequency activity. Recycling of synaptically released glycine is crucial for reliable inhibitory transmission at MNTB LSO synapses. Glycine transporter 2 (GlyT2) mediates glycine (re)uptake into MNTB axon terminals. Glycine is (re)filled into the synaptic vesicles (SV) with the vesicular inhibitory amino acid transporter (VIAAT), driven by a proton gradient generated by vacuolar (H+)-ATPase (V-ATPase). Loss of GlyT2 or V ATPase inhibition impairs transmission but does not cause complete failure, suggesting the presence of additional mechanisms supporting glycine (re)uptake and SV (re)filling. Potential candidates are the alanine serine cysteine 1 transporter (Asc 1) glycine (re)uptake and the Na+/H+ exchanger (NHE) for SV (re)filling. However, their contribution to inhibitory transmission at MNTB-LSO synapses under high-frequency activity remains largely unclear. In the first part of my thesis, I investigated the contribution of Asc 1 in wildtype (WT) and GlyT2 knock-out (KO) mice. Immunohistochemistry in the auditory brainstem confirmed Asc-1 expression in both genotypes. Whole-cell patch clamp recordings from LSO neurons were performed while stimulating MNTB axons (Protocol1 and 2). In KO mice, synaptic strength and vesicle replenishment were severely impaired. In WT mice, synaptic transmission remained stable and was only affected after sustained activity. In the second part, I examined synaptic strength during very prolonged activity (Protocol 3) under pharmacological inhibition of GlyT2 and/or Asc-1 in WT. While synaptic transmission and SV replenishment remained reliable under GlyT2 or Asc 1 inhibition, combined blockade led to impaired transmission and reduced SV (re)filling and replenishment. In the third part of my thesis, I investigated SV (re)filling by performing and reanalyzing data under V-ATPase and/or NHE inhibition. Transmission, SV (re)filling and replenishment were reduced. In addition, NHE inhibition affected action potential conduction fidelity, suggesting a non-specific inhibition. Collectively, my results imply that Asc-1 inhibition reduces glycinergic transmission at MNTB LSO synapses. In KO, the remaining transmission is maintained by Asc-1-mediated glycine (re)uptake. This demonstrates that Asc 1, in addition to GlyT2, serves as a key glycine source at MTNB LSO synapses. Inhibition of SV (re)filling reduces glycinergic transmission, highlighting the essential role of V-ATPase in maintaining reliable transmission at MNTB-LSO synapses. The used NHE inhibitor EIPA is not selective for NHE6 and interferes with AP conduction. Due to these unspecific effects, the functional contribution to synaptic performance could not be directly assessed.