Acute and chronic bilirubin encephalopathy (ABE and kernicterus) are serious neurological conditions associated with Jaundice. When plasma free bilirubin is high, it crosses the blood-brain-barrier and causes neuronal damage in the CNS. This investigation focuses on the damage in the brainstem auditory pathway, using the calyx of Held giant glutamatergic synapse, which forms between the anterior ventral cochlear nucleus and the neurons of the medial nucleus of the trapezoid body (MNTB). Hyperbilirubinaemia was induced in CBA/Ca mice (11-20 days old) by a single intraperitoneal injection of bilirubin (0.5 mg/g) and sulfadimethoxine (0.3mg/g). Auditory function was monitored in vivo by auditory brainstem responses (ABR) using click stimulation under anesthesia (Hypnorm/Midazolam/water, ratio 1/1/2: 0.1µl/g intraperitoneally). Electron microscopy (EM) and immunohistochemistry (IHC) for the presynaptic marker VGLUT2 (vesicular glutamate transporter 2) were performed and patch clamp recording in MNTB neurons were made under voltage clamp in order to record miniature excitatory postsynaptic currents (mEPSCs). Evoked responses were recorded, stimulating the presynaptic trapezoid body fibers at the midline, and clamping the MNTB membrane potential at -65mV or +80mV to investigate AMPAR or NMDAR currents, respectively. ABRs were largely abolished at 4h, but they recovered 24h and 5 days after bilirubin injection. IHC showed reduced staining for VGLUT2 in the calyx of Held at both 4h and 24h after injection. An EM study 4 h after bilirubin injection showed two changes at the calyx of Held synapse: first, the terminals were packed with vesicles and second, the presynaptic terminals were detached from the postsynaptic membrane. 24h after bilirubin injection, the presynaptic terminals had rejoined with the postsynaptic membranes, but the calyx of Held synapses were still abnormally overfilled with vesicles. Whole cell experiments validate the damage of the Calyx of Held synapses showing a significant decrease in the amplitude (62.3±3.5 pA n=7 vs control 83.4±7.3 pA n= 6) and in the frequency of spontaneous mEPSCs, (2.5±0.4 Hz n=7 vs control 7.2±1.1 Hz, n=6) that had recovered after 24h (84.4±8.2, vs control 62.3±3.5 pA, frequency 5.4±0.9 Hz vs control 7.2±1.1 Hz, n=4 and 6 respectively). Evoked AMPAR and NMDAR EPSCs showed a decreased amplitude in 4h injected animals (-4.6±0.3nA and 3.5±0.3nA, respectively vs control -6.9 ±1.2 nA and 4.8 ±0.7nA). We conclude that the acute injection of bilirubin causes short-term damage to the auditory system, involving the calyx of Held and provides a good model for further understanding the mechanisms of bilirubin toxicity in disease. This research was funded by Action On Hearing Loss UK