Acute bilirubin toxicity causes hearing loss through presynaptic degeneration at a central glutamatergic synapse

Severe neonatal jaundice causes bilirubin encephalopathy with complications such as ataxia, delayed neurodevelopment and deafness. The hair cells in the cochlea are spared suggesting central damage but the underlying mechanisms for this hearing loss are unknown. Utilising the Gunn rat model of hyperbilirubinemia I investigated the detrimental effect of high levels of bilirubin on the hearing system, using electrophysiological and imaging techniques combined with in vivo and in vitro studies of the auditory brainstem. My studies have focussed on synaptic transmission between the well characterised giant synapse called the calyx of Held and its target, the principal neuron of the Medial Nucleus of the Trapezoid Body (MNTB), as a model synapse. In vivo auditory brainstem responses (ABR) revealed a significant loss of sound-evoked brainstem activity and increased thresholds after homozygous Gunn rats were exposed to elevated bilirubin levels. Extracellular field potential recordings from the MNTB in vitro using multi-electrode arrays showed impaired synaptic transmission. Whole-cell patch-clamp recordings from MNTB neurons confirmed that their electrophysiological properties remain essentially normal. Significantly, synaptic stimulation failed to elicit EPSCs from the giant synapse, with only smaller synaptic inputs being activated in homozygous Gunn rats exposed to elevated bilirubin levels. Multiphoton imaging of dextranrhodamine labelled calyces in living brain slices revealed degeneration of the presynaptic terminals in Gunn rats exposed to high bilirubin. Electron microscopic images confirmed the loss of the presynaptic terminals observed in living tissue with no sign of degeneration in postsynaptic neurons. The use of neuronal nitric oxide synthase antagonist 7-nitroindazole protected the ABRs and the synaptic transmission in the MNTB. This implicates nitric oxide as playing a key role in bilirubin-induced neurodegeneration of the presynaptic terminals. This research demonstrates for the first time that deafness associated with jaundice is fundamentally a neurodegenerative disease and suggests some novel therapeutic strategies.