Acute hyperbilirubinaemia induces presynaptic neurodegeneration at a central glutamatergic synapse

J Physiol 588.23 (2010) pp 4683–4693 4683

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Acute hyperbilirubinaemia induces presynapticneurodegeneration at a central glutamatergic synapse

Martin D. Haustein, David J. Read, Joern R. Steinert, Nadia Pilati, David Dinsdale and Ian D. Forsythe

Neurotoxicity at the Synaptic Interface, MRC Toxicology Unit, University of Leicester, Leicester LE1 9HN, UK

There is a well-established link between hyperbilirubinaemia and hearing loss in paediatrics,but the cellular mechanisms have not been elucidated. Here we used the Gunn rat modelof hyperbilirubinaemia to investigate bilirubin-induced hearing loss. In vivo auditory brain-stem responses revealed that Gunn rats have severe auditory deficits within 18 h of exposureto high bilirubin levels. Using an in vitro preparation of the auditory brainstem from theserats, extracellular multi-electrode array recording from the medial nucleus of the trapezoidbody (MNTB) showed longer latency and decreased amplitude of evoked field potentialsfollowing bilirubin exposure, suggestive of transmission failure at this synaptic relay. Whole-cellpatch-clamp recordings confirmed that the electrophysiological properties of the postsynapticMNTB neurons were unaffected by bilirubin, with no change in action potential waveformsor current–voltage relationships. However, stimulation of the trapezoid body was unableto elicit large calyceal EPSCs in MNTB neurons of hyperbilirubinaemic rats, indicative ofdamage at a presynaptic site. Multi-photon imaging of anterograde-labelled calyceal projectionsrevealed axonal staining and presynaptic profiles around MNTB principal neuron somata.Following induction of hyperbilirubinaemia the giant synapses were largely destroyed. Electronmicroscopy confirmed loss of presynaptic calyceal terminals and supported the electro-physiological evidence for healthy postsynaptic neurons. MNTB neurons express high levelsof neuronal nitric oxide synthase (nNOS). Nitric oxide has been implicated in mechanisms ofbilirubin toxicity elsewhere in the brain, and antagonism of nNOS by 7-nitroindazole protectedhearing during bilirubin exposure. We conclude that bilirubin-induced deafness is caused bydegeneration of excitatory synaptic terminals in the auditory brainstem.

(Resubmitted 17 September 2010; accepted 7 October 2010; first published online 11 October 2010)Corresponding author I. D. Forsythe: Neurotoxicity at the Synaptic Interface, MRC Toxicology Unit, University ofLeicester, Leicester LE1 9HN, UK. Email: [email protected]

Abbreviations ABR, auditory brainstem response; aCSF, artificial cerebrospinal fluid; EM, electron microscopy; MEA,multi-electrode array; MNTB, medial nucleus of the trapezoid body; 7-NI, 7-nitroindazole; sulfa, sulfadimethoxine;nNOS, neuronal nitric oxide synthase; NO, nitric oxide.

Introduction

Jaundice or hyperbilirubinaemia, as the name implies,is the result of elevated serum levels of bilirubin,a degradation product of haemoglobin. Bilirubin isconjugated in the liver so that this more water-solubleform can be excreted in the bile. Neonatal jaundice canbe caused by failure to conjugate free bilirubin (due toenzyme deficiency in the liver), a high load followingerythrocyte damage (haemolysis) or high turnover leadingto bilirubin accumulation. Immaturity and compromisedliver function means that jaundice is common in newborn

babies but causes little lasting harm if monitored andtreated rapidly; phototherapy is often sufficient to lowerthe concentration of free bilirubin. Severe cases ofneonatal jaundice (particularly in premature infants)are associated with deafness (Gerrard, 1952; Rhee et al.1999) and development of cognitive problems such askernicterus (Shapiro et al. 2006) with symptoms includingataxia and epilepsy. Bilirubin causes the degeneration ofPurkinje neurons in the cerebellum (Schutta & Johnson,1969) and basal ganglia (Perlman et al. 1997) consistentwith ataxia and a central site of action (Shapiro et al.2006).

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Our objective was to test the hypothesis that bilirubincontributes to deafness by causing neurodegenerationin the central auditory pathway, by examining synaptictransmission and neuronal function following bilirubinexposure. We have focused on the calyx of Held excitatorysynapse with its target neuron in the medial nucleus of thetrapezoid body (MNTB) (Schneggenburger & Forsythe,2006). This glutamatergic relay synapse is part of thebrainstem auditory pathway involved in sound localisation(Johnston et al. 2010) and both the synapse and its targethave been well characterised.

We have used the Gunn rat model of hyper-bilirubinaemia (Gunn, 1938; Uziel et al. 1983; Shapiro& Nakamura, 2001); this spontaneous mutation wasisolated from a Wistar colony and first recognisedby their yellow coat colour by Charles Gunn.Homozygous (jj) Gunn rats are distinguished fromtheir heterozygous litter mates (Jj) by this yellowdiscolouration. Homozygous Gunn rats lack the liverenzyme uridine-diphosphoglucuronyl-tranferase, thusrendering them unable to efficiently conjugate and excretebilirubin. This results in jaundice within 48 h after birthand the rats stay jaundiced for the rest of their lives.Hyperbilirubinaemia is induced by administration ofa sulfonamide, which displaces bilirubin from serumalbumin binding sites, raising free bilirubin levelssufficiently to allow entry into the brain (Schutta &Johnson, 1969; Rose & Wisniewski, 1979), producinghearing loss (Shapiro, 1988) and ataxia in homozygousGunn rats. The site of action of bilirubin in the auditorysystem is unknown. A peripheral site of action is unlikely,since cochlea hair cells were unaffected (Uziel et al.1983; Rhee et al. 1999; Shaia et al. 2005). Anatomicalpost-mortem studies of humans suffering from severeneonatal jaundice report no pathological lesions in eitherthe cochlea or dorsal root ganglia but extensive destructionof neurons in the cochlear nucleus (Dublin, 1951).Sound-evoked auditory brainstem responses (ABRs) arecommonly used to test for hearing loss and hyper-bilirubinaemia reduces ABRs in human neonates (Chisinet al. 1979) and homozygous Gunn rats (Shapiro, 1988).Functional hearing tests in homozygous Gunn rats suggestthat the failure of the ABR is associated with a reducedsize of the cochlear nuclei and the MNTB (Conlee &Shapiro, 1991) but the underlying cellular defect has notbeen identified.

Our results show reduced ABR amplitudes indicatinghearing loss after 18 h hyperbilirubinaemia. Intra- andextracellular electrophysiological recordings demonstratethat this was accompanied by failure of synaptic trans-mission at the calyx of Held/MNTB synapse. Multi-photonimaging and electron microscopy reveal substantialdegeneration of the presynaptic terminal, while thepostsynaptic MNTB neurons remain largely unaffected,

confirming a presynaptic site of action for bilirubindamage.

Methods

Ethical approval

All experiments were performed at the MRC ToxicologyUnit in Leicester (UK), in accordance with the UK Animals(Scientific Procedures) Act 1986 and under the approval ofthe local ethical committee of the University of Leicester.All experiments reported in this study comply with thepolicies and regulations of The Journal of Physiology(Drummond, 2009).

A total of 73 Wistar and Gunn rats (Harlan UK, strainHsdBlu:GUNN-UDPGT j) aged from postnatal day 14 to20 (P14–20) were used because blood bilirubin levelsnaturally peak in homozygous Gunn rats at between 2and 3 weeks of life (Schutta & Johnson, 1969). Animalswere bred in-house and homozygous (jj) Gunn rats wereobtained by mating jj male with heterozygous (Jj) femaleGunn rats (Takagishi & Yamamura, 1994).

Induction of hyperbilirubinaemia

Separate studies from Wistar, Jj or jj Gunn rats showedsimilar electrophysiological properties under whole-cellpatch and multi-electrode array (MEA) recording andwere pooled for later experiments as the control group(control). The intraperitoneal (I.P.) administration of thelong-lasting sulfonamide sulfadimethoxine (sulfa; Rose &Wisniewski, 1979; Shapiro, 1988) (200 mg kg−1; Sigma)was used to trigger hyperbilirubinaemia in jj-Gunn rats.The experiments were conducted 18 h after this treatment(treated group). Prior to the animals being killed and 18 hfollowing sulfa treatment, 77% of jj-Gunn rats showedmotor deficits or ataxia. All treated jj-Gunn rats thathad motor deficits also showed compromised synaptictransmission or degenerated/absent calyces. Wild-typeWistar rats that received identical 18 h sulfa treatmentwere asymptomatic, showing no motor deficits, ataxia, nocompromised ABRs (Fig. 4C) or impaired synaptic trans-mission in the MNTB. Similar results from Jj-Gunn ratshave been reported (Rose & Wisniewski, 1979; Shapiro,1988).

Auditory brainstem responses (ABRs)

Homozygous Gunn rats and Wistar rats were anaesthetisedwith an I.P. injection of Hypnorm/midazolam(2.7 ml kg−1, VetaPharma Ltd) and recordings were madebefore (control) and 18 h after (treated) administration ofsulfadimethoxine (200 mg kg−1, Sigma) using a MedelecSapphire 2A amplifier. Data were sampled at 16 kHz

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and stored on a personal computer. ABRs were evokedusing pure-tone (12, 24, 30 kHz) and ‘click’ stimulidelivered via a reverse-driven microphone (B&K 4134)over a range of intensities (24–94 dB) using a digitalattenuator (Tucker Davies Technologies, USA). For someexperiments an nNOS antagonist, 7-nitroindazole (7-NI,Sigma) was suspended in peanut oil and administered I.P.at a concentration of 150 mg kg−1, 30 min prior to the I.P.injection of sulfadimethoxine.

Patch clamp

Brain slices were prepared from animals killed bydecapitation. Whole-cell patch recordings were madefrom visually identified rat MNTB neurons in acutebrain slices (200 μm thick) of the auditory brain-stem as described previously (Steinert et al. 2008).Experiments were performed at 36 ± 1◦C using afeedback-controlled Peltier device (manufactured byUniversity of Leicester Mechanical and ElectricalWorkshop). Synaptic stimulation (using a DS2A isolatedstimulator (Digitimer, Welwyn Garden City, UK; 1–10 V,0.1–0.2 ms) was delivered via a bipolar platinumelectrode placed at the midline across the trapezoidbody nerve fibres. Patch pipettes were pulled fromglass capillaries (GC150F-7.5, o.d. 1.5 mm, HarvardApparatus, Edenbridge, UK) and had resistances of2–5 M� when filled with the pipette solution (see below).Series resistances were between 4 and 9 M� (seriesresistance compensation and prediction were around70%). Data were recorded using a Multiclamp 700Bamplifier (Molecular Devices, Sunnyvale, CA, USA).Stimulation, data acquisition and analysis were performedusing pCLAMP 9 and Clampfit 10.2 (Molecular Devices).Average data are presented as mean ± S.E.M.

Calcium imaging

Acute brainstem slices (200 μm) were prepared as forpatch clamp experiments. Slices were loaded with 5 μM

of the acetoxy methyl ester form of Fura-2 (Fura-2 AM;Molecular Probes, Eugene, OR, USA, dissolved inDMSO with 5% pluronic acid) for 10 min, thenmaintained for 30 min in artificial cerebrospinal fluid(aCSF) for de-esterification of the AM-dye. Fura-2fluorescence (excitation at 340 and 380 nm) was recordedand Metafluor imaging software (Series 7, MolecularDevices) was used to display the fluorescence images(emission > 505 nm).

Extracellular multi-electrode array

Acute brainstem slices (300 μm thick) were preparedas described above. One slice was placed on top of

a multi-electrode array dish (MED-P515A, 150 μmelectrode spacing) and placed in a recording chamber(MED64, Alpha MED Scientific Inc., Japan), which wasadapted for perfusion and Peltier-driven temperaturecontrol (manufactured by University of LeicesterMechanical and Electrical Workshop). Stimulation witha biphasic pulse (±100 μA, 160 μs) to one or twoelectrodes at the midline was used to evoke field potentialsin the superior olivary complex. Evoked MNTB fieldpotentials (Haustein et al. 2008) were recorded frombetween 4 and 10 underlying electrodes. Data wererecorded at a sampling rate of 20 kHz with 12 bitresolution. Extracellular recordings were performed at31 ± 1◦C using Mobius software (Alpha MED ScientificInc.) for stimulation, recording and data analysis.

Multi-photon live imaging

Calyces in the MNTB were anterogradely labelledby injection of dextran–tetramethyl-rhodamine (MW3000; Invitrogen) in vitro into the ventral cochlearnucleus using methods adapted from Burger et al.(2005). Dextran–rhodamine (5% in sterile PBS) waspressure-injected and electroporation via a bipolartungsten electrode connected to a BTX ECM 830 electro-porator (Harvard Apparatus; 1 × 120 V pulse for 130 ms,then 60 × 50 ms 50 V pulses) was employed to induceuptake of the dye into the bushy cells that give rise to thecalyces of Held. Slices were then kept in the dark at roomtemperature in a maintenance chamber for at least 4 hbefore imaging. Images and z-stacks from the living slicewere taken using a Zeiss LSM510 scanning microscopeequipped with a Mai-Tai Deep-See multi-photon laser(Spectra-Physics). Images were analysed and the surfacearea of calyces was calculated from z-stacks using Volocity5.3 software (Improvision, UK). The calyces were visuallyidentified by their shape. Great care was applied to onlymeasure the calyces individually and to avoid adjacent oroverlapping structures, such as neighbouring calyces oraxons.

Electron microscopy

Rats were anaesthetised with isofluorane (3–4%) andthen perfusion-fixed with 2% glutaraldehyde + 2%paraformaldehyde in 0.1 M sodium cacodylate buffer (finalpH 7.4). Slices (500 μm thick) were prepared using avibrating blade microtome (Leica Microsystems, MiltonKeynes, UK). These slices were post-fixed in 1% osmiumtetroxide + 1% potassium ferrocyanide, stained en blocwith 5% uranyl acetate and embedded in epoxy resin(TAAB Laboratories Equipment Ltd, Aldermaston, UK).Semi-thin (1 μm) sections were stained with toluidineblue and examined to select areas for ultramicrotomy.

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Ultrathin sections (70 nm) were stained with leadcitrate and examined in a JEOL 100-CXII electronmicroscope (JEOL (UK) Ltd, Welwyn Garden City, UK)equipped with a ‘Megaview III’ digital camera (OlympusSoft Imaging Solutions GmbH, Munster, Germany).Analysis of electron microscopy (EM) images was donedouble-blinded using ImageJ 1.42 (NIH) or Axiovision4.8 software (Zeiss). Presynaptic profiles were identifiedas structures enclosed by a membrane in contact with thecell body, showing a postsynaptic density and containingsynaptic vesicles. Neurons containing the principal cellnucleus were selected for this analysis.

Solutions

An artificial cerebrospinal fluid (aCSF) was used forslice incubation, maintenance after slicing and perfusionduring recordings (in mM): NaCl 125, KCl 2.5, NaHCO3

26, glucose 10, NaH2PO4 1.25, sodium pyruvate 2,myo-inositol 3, CaCl2 2, MgCl2 1, ascorbic acid 0.5; pHwas 7.4 when gassed with 95% O2, 5% CO2. Osmolaritywas around 310 mosmol l−1. A low-sodium aCSF was usedduring preparation of slices, with a composition as abovefor aCSF, except that NaCl was replaced by sucrose 250 mM,and CaCl2 and MgCl2 were changed to 0.1 mM and 4 mM,respectively. The pipette solution for whole-cell recordingscontained (in mM): potassium gluconate 97.5; KCl 32.5,Hepes 10, EGTA 5, MgCl2 1; pH was adjusted to pH7.3 with KOH and osmolarity to 290 mosmol l−1 withsucrose.

Numbers and statistics

The number (n) for statistical analysis depended on theexperimental design (i.e. animals used in vivo; the numberof cells where definable or the number of independentelectrodes for MEA recordings). A summary of the animalsused is as follows:(1) ABR. 8 jj-Gunn rats (control), 7 rats (sulfa-treated), 2rats (saline), 4 Wistar rats (sulfa), 7 jj-Gunn rats (7-NI);each animal served as its own control (ABR only).(2) MEA. 52 electrodes/6 slices/3 rats (control), 55 electro-des/9 slices/4 rats (treated).(3) Synaptic stimulation and voltage clamp. 15 cells/7rats (control), 9 cells/5 rats (treated).(4) Voltage clamp. 9 cells/3 rats (control), 18 cells/3 rats(treated).(5) Current clamp. 26 cells/10 rats (control), 18 cells/7rats (treated).(6) Multi-photon imaging. For box-plot summary: 20calyces/3 rats (control), 13 calyceal remnants/2 rats(treated).(7) Electron microscopy. 24 cells/3 rats (control), 18cells/2 rats (treated).

The specific statistical test is noted in the text or therespective figure legend, which was performed using SigmaPlot 11 (Systat Software Inc.). Statistical significance wasaccepted if P < 0.05.

Results

Control untreated Gunn rats aged P16–P18 showednormal in vivo ABR responses, but following 18 h of hyper-bilirubinaemia (Fig. 1A) the characteristic ABR waveformswere severely disrupted, consistent with damage to thecentral auditory system (Shapiro, 1988). Wave III of theABR, which is thought to reflect activity in the superiorolivary complex (Melcher et al. 1996) was significantlyreduced in amplitude in all treated jj-Gunn rats (n = 7,Fig. 1A and B). Two animals which received saline insteadof sulfadimethoxine showed no change in ABR response(data not shown). Extracellular MEA recordings frombrainstem slices in vitro (Fig. 1C) showed a characteristicpresynaptic conducted waveform on stimulation of thetrapezoid body (C1) and a postsynaptic component (C2)(Haustein et al. 2008). In treated rats the C2 componentwas absent or vastly reduced (control −186.4 ± 18.7 μV,n = 52; vs. treated −32.1 ± 3.2 μV, n = 55; P < 0.001,Mann–Whitney-Rank-Sum test) as shown by the C2/C1

ratio for both conditions (Fig. 1D; P < 0.001). The MEAdata also revealed an increased synaptic delay, from0.46 ± 0.01 ms in control (n = 52) rising to 1.12 ± 0.03 msin animals with hyperbilirubinaemia (treated, n = 55,P < 0.001, Mann–Whitney-Rank-Sum test). In order toconfirm the link between the ABR and MEA data, we madebrain slices from three treated jj-Gunn rats. Each showedreduced ABRs in vivo, and the postsynaptic C2 componentin vitro was either absent or reduced in amplitude andexhibited increased synaptic delay. Clearly compromisedABRs in hyperbilirubinaemia reflected impaired synaptictransmission in the MNTB.

We performed whole-cell patch recording from MNTBprincipal neurons in the same in vitro preparation.Treated jj-Gunn rats showed no large amplitude evokedEPSCs, with mean amplitudes of 1.5 ± 0.5 nA (n = 9)compared with control EPSCs of 9.6 ± 1.2 nA (n = 9, at−60 mV). At higher stimulus intensities (>6 V, Fig. 2A)non-calyceal EPSCs were observed (Hamann et al. 2003) intreated Gunn rats. The input/output plot showed controlcalyceal EPSCs as low threshold and large magnitudeevents (Fig. 2A) while responses from treated animalswere of high threshold and low magnitude (Fig. 2A). Nodifference was observed in excitability or action potentialwaveform between MNTB neurons from control andtreated animals (Fig. 2B) nor were inward sodium currents(control 13 ± 1 nA, n = 9; vs. treated 12 ± 1 nA, n = 18;at −100 mV) or outward potassium currents affected(Fig. 2C). These results show that 18 h elevated bilirubin

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causes a rapid failure of synaptic transmission withoutaffecting postsynaptic excitability.

The electrophysiological data indicate a presynapticfailure of synaptic transmission, so we performedimaging experiments at the light and EM level to testfor presynaptic degeneration. The presynaptic axonsand calyces of Held were anterogradely labelled withdextran–rhodamine and live imaging performed usingmulti-photon microscopy, in vitro. Control rats showedwell-labelled calyceal structures in the MNTB (Fig. 3A)but 18 h after induction of hyperbilirubinaemia nohealthy calyces were observed (Fig. 3B). The surfacearea (2334 ± 301 μm2, n = 20) of healthy calyces wasconsistent with previous reports (Satzler et al. 2002), whilecalyceal remnants from treated animals (1117 ± 318 μm2,n = 13, P > 0.001; Mann–Whitney-Rank-Sum test) weresignificantly smaller and there were considerable numbersof isolated dye-containing structures, indicating calycealbreakdown (Fig. 3B, inset). These remnants were excludedfrom this analysis, and so the above mean areas under-estimate the total degree of synaptic breakdown. Intensely

stained axons were present in both conditions (Fig. 3A andB inset), consistent with intact axons and functional dyetransport.

Electron-micrographs of the MNTB confirmed thepresynaptic pathology; controls showed the targetprincipal neuron surrounded by multiple large pre-synaptic calyceal profiles containing vesicles andmitochondria (yellow overlay, Fig. 3C). In treatedanimals the MNTB neuron looked healthy, but closerexamination showed reduction in the number of pre-synaptic profiles (control 11 ± 1, n = 24; vs. treated 7 ± 1,n = 18; P > 0.006; Fig. 3D and F) and their length ofapposition to the target neuron (control 23 ± 2 μm, vs.treated 14 ± 2 μm; P > 0.006; Fig. 3E). Principal neuronperimeters were unaffected by bilirubin treatment(control 68 ± 2 μm, vs. treated 74 ± 3 μm; not significantFig. 3E). Mitochondria and endoplasmic reticulum (ER)showed no pathology, consistent with minimal post-synaptic excitotoxicity. An uncompromised, healthy post-synaptic neuron was also confirmed by Ca2+ imagingwhich showed no elevation of resting [Ca2+]i in MNTB

Figure 1. Jaundice causes hearing loss through failure of transmission in the auditory brainstemA, in vivo auditory brainstem responses (ABRs) exhibit well-defined waves (I–IV) in response to 30 kHz pure-toneat 94 dB (black trace, control, n = 8); hyperbilirubinaemia in the jj-Gunn rat (grey trace, treated, n = 7) causedsevere suppression of waves I–III within 18 h. B, bar graph shows that the mean amplitudes of ABR waves I–III aresignificantly reduced after 18 h hyperbilirubinaemia. t test, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. C, extracellularfield potentials from the MNTB in vitro show a presynaptic (C1) and postsynaptic (C2) component evoked bystimulation of the trapezoid axons (control, black trace); C1 is unaffected while C2 is delayed and suppressed byhyperbilirubinaemia (grey trace; s, stimulation artefact). D, bar graph shows mean reduction of C2 amplitude intreated animals. All values are mean ± S.E.M.; ANOVA, ∗∗∗P < 0.001.

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neurons between control (110 ± 7 nM, n = 71) and treated(111 ± 10 nM, n = 46) Gunn rats.

Why is this synapse so susceptible to bilirubin toxicity?Recent reports (Brito et al. 2010) have implicatednitric oxide (NO) in bilirubin toxicity. Previously wehave shown that the MNTB expresses high levels

of neuronal nitric oxide synthase (nNOS) (Steinertet al. 2008), so we reasoned that an nNOS antagonist,7-nitroindazole (7-NI) might protect hearing in thisGunn rat model. Administration of 7-NI during bilirubinexposure provided significant protection from loss ofauditory function (Fig. 4). This was quantified by using

Figure 2. Patch-clamp recording reveals that principal neurons exhibit normal excitability, but the giantcalyceal EPSC is absent following hyperbilirubinaemiaA, under voltage clamp MNTB neurons normally receive a large calyceal EPSC (black trace) but only small slow EPSCsare present following hyperbilirubinaemia (grey trace). A plot of EPSC amplitude against threshold stimulus intensityshows that control calyceal EPSCs (black diamonds) were low threshold and large amplitude (grey, highlighted area),whereas the EPSCs from treated animals (grey squares) were small amplitude and high threshold. B, postsynapticMNTB neuron excitability was unchanged by hyperbilirubinaemia, as indicated by the similar action potential (AP)waveforms: although AP halfwidth was similar, there was a small increase in AP amplitude in treated animals.C, control (black) and treated (grey) principal neurons show near identical current–voltage relationships. Insetsshow example current traces from one cell, with voltage commands plotted below. All values are mean ± S.E.M.;Mann–Whitney-Rank-Sum test; n.s., not significant; ∗P < 0.05.

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the threshold to a ‘click’ sound stimulus. Wistar and Gunnrats had similar thresholds of around 40 dB under controlconditions but 18 h after induction of hyperbilirubinaemiathe jj-Gunn rats’ thresholds were raised to 80 dB (Fig. 4C);this threshold elevation was substantially prevented whenthe jj-Gunn rats received the nNOS antagonist 7-NI. Theseresults suggest that the mechanism of bilirubin toxicityinvolves pathological changes in the nitrergic signalling ofthe MNTB.

Discussion

ABR measurements reveal substantial hearing loss inGunn rats within 18 h of exposure to high bilirubin.Extracellular multi-electrode array recordings showedimpaired synaptic transmission through the MNTB invitro. Whole-cell patch-clamp recordings from MNTBneurons in hyperbilirubinaemic rats confirmed thattheir electrophysiological properties were essentiallyunchanged from control animals. However, stimulationof the trapezoid body was unable to elicit large amplitudecalyceal EPSCs in MNTB neurons of hyperbilirubinaemicGunn rats. Multi-photon imaging of anterogradelylabelled calyceal terminals revealed dramatic degenerationof the presynaptic calyx, supporting a neurodegenerativemechanism. Electron microscopy confirmed the loss ofpresynaptic terminals and healthy postsynaptic neurons.The protection from hearing loss by an nNOS antagonistsuggests involvement of nitric oxide signalling in this pre-synaptic toxicity. We conclude that degeneration underliesthe synaptic failure in the MNTB, resulting in reduced ABRamplitudes in an acute model of hyperbilirubinaemia.

Following 18 h hyperbilirubinaemia (induced by sulfaadministration) we observed decreased amplitudes orcomplete absence of ABR waves I–III. These ABR data arein accordance with findings in homozygous Gunn rats byShapiro (1988) where declining amplitude and increasedwaveform latency occur between 1 and 8 h after inductionof hyperbilirubinaemia. Acute in vitro slices obtained fromanimals which had undergone in vivo ABR measurementsconfirmed a common defect. Field potentials measuredfrom treated Gunn rats showed prolonged synapticdelays and decreased amplitudes of the postsynaptic fieldpotential (C2). The MNTB contributes to the generationof waveform III in ABR recordings (Melcher et al. 1996);we can therefore directly link the impaired synaptictransmission measured with MEAs to the reduced wave-form III amplitude indicative of hearing loss. The MEAC2 synaptic latency matched that of in vivo recordings(0.46 ± 0.12 ms) (Kopp-Scheinpflug et al. 2003). Inter-estingly, the prolonged synaptic delay measured here intreated jj-Gunn rats (1.12 ± 0.03 ms) is similar to thatreported for the endbulb of Held/ventral cochlear nucleusof treated jj-Gunn rats (1.15 ± 0.23 ms Zhang et al. 1989),

consistent with an early presynaptic compromise causingincreased synaptic delay. MEA data did not providesufficient information on the source of transmissionfailure in the MNTB so we performed patch-clamprecordings on single MNTB neurons to address thisquestion. Whole-cell patch-clamp recordings from treatedMNTB neurons were unaffected by bilirubin and the post-synaptic neuron did not degenerate in this acute model ofhyperbilirubinaemia. This argument was strengthened bymeasurement of resting [Ca2+]i levels which were withinnormal ranges. However, low-threshold, large amplitudecalyceal EPSCs could not be evoked in any of the MNTBneurons from treated Gunn rats. In contrast, neurons fromcontrol rats commonly showed calyceal EPSCs. Bilirubinhas no direct effect on postsynaptic ionotropic glutamatereceptors (Warr et al. 2000), so together these data suggesta presynaptic rather than postsynaptic site of failure insynaptic transmission.

Multi-photon imaging of labelled calyces of Held inacute brain slices from treated rats revealed degeneratedor completely absent calyces. Axons were clearly labelledin both conditions indicating functional transport ofdextran–tetramethyl-rhodamine from the bushy cellbodies of the anteroventral cochlear nucleus to the calycesof Held. While calyces in control animals had the samemorphology as previously described (Forsythe, 1994),treated animals showed little intact calyceal structure anda large amount of stained debris, where calyces wouldhave been expected. These remnants are probably parts ofdisintegrated calyces. While other authors have describeda decrease in cell size of MNTB neurons (Conlee &Shapiro, 1991) after 4–5 days of sulfa treatment, anda reduction in the expression of the calcium-bindingprotein parvalbumin (Spencer et al. 2002) after 3 days ofsulfa treatment, no previous studies have investigated orcommented on the structural integrity of the presynapticterminals. Shaia and colleagues reported degenerativechanges in spiral ganglion neurons of jj-Gunn rats after3 days of sulfa treatment (Shaia et al. 2005) althoughcochlear hair cells were not affected.

There is EM evidence from sulfa-treated Gunn rats thatboth acute lesions as well as chronic degeneration occursin the cerebellar Purkinje cells in this model of hyper-bilirubinaemia (Rose & Wisniewski, 1979). At 18 h theacute effects of bilirubin observed in this study caused nomeasurable change in MNTB neurones. Darkening of theremaining MNTB presynaptic profiles in treated Gunn ratsmatches observations in the cochlear nuclei of Gunn rats(Jew & Williams, 1977; Jew & Sandquist, 1979), althoughother hallmarks of neurodegeneration such as distortedmitochondria, accumulation of mitochondrial glycogenand abnormally increased extracellular space were notobserved in the MNTB. In contrast, postsynaptic diseasemechanisms seem more prevalent in the cerebellum ofjj-Gunn rats which has long been known to be affected

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Figure 3. Hyperbilirubinaemia causes degeneration of the calyceal presynaptic terminalA, multi-photon imaging of live calyces (labelled with dextran–rhodamine, MW 3000) from a control rat (P14,jj-Gunn, untreated) showing axons and 3 calyceal synaptic structures. Inset: comparison of the calyx surface areasbetween control and treated animals shows a significantly reduced surface area in the degenerating calyces.Mann–Whitney-Rank-Sum test, ∗∗∗P < 0.001. B, 18 h following hyperbilirubinaemia, no healthy calyces wereobserved, but considerable labelled debris was present; inset: an example of a rare calyx remnant with markeddegenerative morphology. Scale bars in A, B and inset: 20 μm. C, electron microscopy confirms the presence ofthe calyceal presynaptic terminal, showing a control MNTB neuron, surrounded by presynaptic terminal profiles(yellow). Mitochondria, ER and myelinated axon profiles are clearly visible. D, hyperbilirubinaemia reduced the

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Figure 4. Protection from bilirubin-induced hearing loss by the nNOS inhibitor, 7-nitroindazole (7-NI)A, ABRs from a jj-Gunn rat in response to a 94 dB ‘click’ stimulus before (black) and after (grey) induction ofhyperbilirubinaemia (18 h after sulfadimethoxine, 200 mg kg−1). B, ABRs from a jj-Gunn rat in response to a94 dB ‘click’ stimulus before (black) and after (grey) induction of hyperbilirubinaemia (18 h after sulfadimethoxine,200 mg kg−1, and 7-NI, 150 mg kg−1). C, summary plot of ABR thresholds in response to a ‘click’ stimulus.Wild-type Wistar rats and the jj-Gunn rats have similar auditory thresholds under control conditions. The Wistar ratssuffered no auditory deficit after 18 h sulfadimethoxine, whereas jj-Gunn rats show significantly elevated thresholdsafter sulfa treatment. 7-NI protected the hearing from this threshold elevation. Box plots show mean ± upper andlower threshold range; ANOVA, ∗P < 0.05. The number of animals tested is indicated in the respective box.

in hyperbilirubinaemia (Schutta & Johnson, 1969; Rose& Wisniewski, 1979). The sensitivity of the excitatorysynapses in the auditory brainstem to bilirubin (Shapiro& Nakamura, 2001) probably explains the association ofneonatal jaundice with deafness, and suggests that othercognitive and motor deficits associated with jaundice havesimilar underlying mechanisms, e.g. in the basal ganglia,cerebellum and hippocampus (Shapiro et al. 2006).

Human neonates suffering from bilirubinencephalopathy/kernicterus also show light micro-scopic evidence of bilirubin-induced neurodegenerationwith lesions in the cochlear nuclei, with pyknosis anddisintegration (Gerrard, 1952). The cochleae, 8th nerveand spiral ganglion neurons did not show abnormalities.In an autopsy study (Dublin, 1951) reduced numbersof fibres in the trapezoid body were reported, implyingfewer synapses, consistent with the present data fromthe Gunn rat model. In a recent autopsy case study,

Perlman and colleagues also report necrosis in thehippocampus (Perlman et al. 1997) following kernicterus.These studies emphasise the particular vulnerability ofthe auditory system and basal ganglia and highlight theneed for more research in animal models (such as theGunn rat) to unravel the underlying causes of bilirubinneurotoxicity and mitigate the long-term detrimentaleffects of bilirubin-induced neurodegeneration.

Previous reports have suggested that injection ofalbumin causes some reversal of bilirubin toxicity,presumably by binding free bilirubin (Shapiro, 1993). Interms of an underlying mechanism, it has been suggestedthat antagonists for NMDA receptors (McDonald et al.1998) or nNOS (Brito et al. 2010) can provide protectionfrom bilirubin toxicity. The MNTB has high levels ofnNOS (Steinert et al. 2008) and we show here thatan nNOS antagonist provides substantial protection ofhearing from bilirubin-induced toxicity. Further work is

number of presynaptic profiles (yellow), but the principal neuron and internal organelles appeared otherwisenormal. E and F, MNTB neuron perimeter was unchanged, but the length and number of presynaptic profiles weresignificantly reduced by hyperbilirubinaemia, confirming the hypothesis of presynaptic degeneration. t test; n.s.,not significant; ∗∗P < 0.01. Scale bars in C and D: 2 μm.

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4692 M. D. Haustein and others J Physiol 588.23

required to identify the mechanism by which bilirubincauses NO production, and to establish the downstreamactions of nitric oxide and mechanisms of toxicity (Steinertet al. 2010). We conclude that hyperbilirubinaemia causesdegeneration of excitatory synaptic terminals in theauditory brainstem and this is associated with activationof neuronal nitric oxide synthase. Further investigation isrequired to test the therapeutic and wider implications ofthis result to other areas of the nervous system.

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Author contributions

M.D.H. conducted the experiments and analysis. D.J.R. providedexpert assistance and helped with analysis of multi-photon

imaging and EM data. J.R.S. conducted the calcium-imagingexperiments. N.P. performed additional ABR recordings. D.D.conducted the EM imaging. I.D.F. developed the experimentaldesign, interpreted data and supervised the project. M.D.H. andI.D.F. wrote the manuscript. All authors provided discussion,comments and approved the manuscript.

Acknowledgements

We would like to thank Judy McWilliam and Tim Smith for theirhelp in preparing samples for electron microscopy, Robert Fernfor advice on CNS pathology and Brian Robertson for drawingour attention to this fascinating problem. This work was fundedby the MRC and by a PhD Scholarship to M.D.H. from DeafnessResearch UK.

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