Will you please listen to my ear? There must be something in there, I fear

Richard Rabbit

Professor of Bioengineering, Adjunct Professor of Otolaryngology, University of Utah, USA

The remarkable sensitivity of auditory and vestibular organs is achieved through active mechanical amplification by sensory hair cells. Amplification of low strength stimuli, but not high strength stimuli, results in compression that maps a wide dynamic range of stimuli into a smaller range of micromechanical hair bundle displacements. Compression is present in both auditory and vestibular organs, and is exceptionally large in the mammalian cochlea. Cochlear amplification is controlled by the brain through centrifugal efferent projections that, when activated, reduce the gain of the outer hair cell electromotility and sharply attenuate the vibration of the cochlear partition. This centrally controlled strategy is essential to the exquisite sense of hearing in mammals. We recently demonstrated that a similar active process is present in vestibular organs, even though they lack outer hair cells and the protein prestin. We measured micromechanical motion of the semicircular canal cupula overlying hair bundles as a function of stimulus level and discovered that low-level stimuli were amplified resulting in larger cupula motions than would occur in a passive system. Like the cochlea, amplification in the semicircular canals was eliminated by electrical activation of the brainstem efferent vestibular nucleus. In the presentation, I will compare and contrast amplification by semicircular canal hair cells to amplification by cochlear outer hair cells to reveal general principles used by the ear to achieve sensitivity near the level of thermal noise. (Funded by the US National Institutes of Health R01 DC006685)

Trait Anxiety Affects the Development of Tinnitus Following Acoustic Trauma

Paolini, A.G.1,2,3, Pizzica, S. E1., Samuell, A. V1., Allitt, B. J.4, Kim J.3 and Mulders W.H.A.M5

1 2 3 School of Health Sciences, RMIT University; School of Psychological Science, ; The 4 5 Florey Institute of Neuroscience and Mental Health; Department of Physiology, Monash University; School of Anatomy, Physiology and Human Biology, University of Western

There are inconsistencies in theory surrounding the mechanisms involved in the development tinnitus. This research aimed to assess the influence of anxiety levels on tinnitus development in rats. Anxiety levels, tinnitus development and sensorineural hearing loss were assessed in twenty two 8-12 week male Wistar rats. Rats were placed in an elevated plus maze (EPM) to assess trait anxiety levels. Four days after EPM protocols, sensorineural hearing loss (NHL) was induced in 14 animals using high amplitude octave band-pass filtered noise (peak intensity ranging between 108 dB –120 dB SPL at 16 kHz). 8 Animals underwent the same procedure without noise exposure and acted as a control. At seven days and fifteen days after NHL/SHAM, animals were exposed to gap pre-pulse inhibition acoustic startle (GPIAS) testing to assess the severity of induced tinnitus. Following behavioural protocols, animals were anaesthetised (2.6g/kg urethane ip) and placed in a stereotaxic frame and Pure tone bursts (1-44 kHz, 20-80 dB SPL, 15 reps of each frequency/intensity combination) were presented to each ear while recording from a 32 channel array inserted in each inferior colliculus and in the lateral amygdala.

Cohen’s anxiety measure (1) was calculated via EPM data and used as the criteria for separating rats into high anxiety (HA) and low anxiety (LA) groups. The level of tinnitus was determined by taking the mean peak-peak startle amplitude of the gap and startle trials for each noise condition (2,3). The tinnitus score calculated was the ratio of the mean peak-peak startle amplitudes for the gap trials to the mean peak-peak startle amplitudes of the startle trials.

Rats with elevated levels of anxiety had a greater propensity to develop tinnitus in comparison to rats with lower anxiety. There was a significant interaction between anxiety and tinnitus scores in the GPIAS test using 10 kHz broadband filtered noise between seven and 15 days post NHL. Post-hoc comparisons show that the HA group mean tinnitus scores was significantly higher at 15 days compared to seven (t = 2.88, p = .01) and significantly higher than the LA group tinnitus score at 15 days (t = 2.62, p = .02). There was a significant positive correlation between anxiety score and tinnitus score 15 days post NHL in the 10kHz octave band- pass filtered noise condition (r = .74, p < .01). This was not the case with white noise nor was this relationship seen in control animals at any on the frequencies. Electrophysiologically there was a significant elevation in spontaneous activity in the lateral amygdala of HA animals, together with elevation in spontaneous activity of multiunit clusters at the frequency edge of the leasioned frequency regions. This is not the case for animals not displaying tinnitus and having low anxiety. Our preliminary results strongly suggest that pre-existing anxiety has an effect on the development and maintenance of tinnitus reflected through changes in neural excitability. 1.Cohen H, Liu T, Kozlovsky N, Kaplan Z, Zohar J, Mathe AA. The neuropeptide Y (NPY)-ergic system is associated with behavioral resilience to stress exposure in an animal model of post-traumatic stress disorder. Neuropsychopharmacology 2012;37(2):350-63. 2. Turner JG, Brozoski TJ, Bauer CA, Parrish JL, Myers K, Hughes LF, et al. Gap detection deficits in rats with tinnitus: a potential novel screening tool. Behavioral neuroscience. 2006;120(1):188-95. 3. Pace E, Zhang J. Noise-induced tinnitus using individualized gap detection analysis and its relationship with hyperacusis, anxiety, and spatial cognition. PloS one. 2013;8(9):e75011. Characterisation of Cochlear Inflammation in Mice after Acute and Chronic Noise Exposures and Potential Treatment

Tan W.J.T.1,2, Telang R.S.1,2, Thorne P.R.1,2,3, Vlajkovic S.M.1,2

1Department of Physiology, 2Centre for Brain Research, 3Section of Audiology, The University of Auckland, Auckland, New Zealand

Previous studies have shown that exposure to excessive noise induces an inflammatory response in the cochlea which may contribute to noise-induced cochlear injury and hearing loss. This study aimed to improve our understanding of the underlying mechanisms and dynamics of the noise- induced inflammatory response in the mouse cochlea. To investigate the inflammation with acute noise exposure, cochleae were collected from adult (6-8 weeks) C57BL/6 mice at various intervals (6 h to 7 days) after acute exposure to traumatic noise (100 dB SPL, 8-16 kHz, 24 h). Using quantitative real-time RT-PCR and immunohistochemistry, noise-induced changes in expression levels of proinflammatory cytokines (TNF-α, IL-1β), chemokines (CCL2) and cell adhesion molecules (ICAM-1) were studied. All gene transcripts displayed similar dynamics of expression, with an early upregulation at 6 h post-exposure, followed by a second peak at 7 days. ICAM-1 immunoexpression in the spiral ligament fibrocytes and endothelial cells increased significantly, peaking at 24 h post-exposure. We also demonstrated the presence of adenosine A2A receptor

(A2AR)-positive cells in the cochlea, most likely immune cells recruited from the circulation and with a maximum infiltration observed at 24 h. These findings suggest that cytokines and cell adhesion molecules initiate noise-induced cochlear inflammation and mediate the recruitment and extravasation of inflammatory cells. We speculate that these cells are recruited to clear cellular debris, but may also cause significant bystander tissue injury, thus exacerbating noise- induced hearing loss. The occurrence of the latter peak in expression of inflammatory mediators is not clear, but we postulate that it may be associated with reparative processes. In a further study, chronic exposure to moderate noise levels (90 dB SPL, 8-16 kHz, 2 h/day, up to 4 weeks) also induced an inflammatory response, peaking after 2 weeks, and subsiding thereafter, suggesting that inflammation may contribute to cochlear injury with chronic noise exposure even at moderate sound levels. Furthermore, we demonstrated that post-exposure treatment with regadenoson, a FDA-approved selective A2AR agonist, suppressed cochlear inflammation, by significantly reducing ICAM-1 immmunoexpression and cellular infiltration. This reveals an important role of A2AR signalling in controlling noise-induced cochlear inflammation, and pinpoints

A2AR as an attractive pharmacological target in this condition.

This study was supported by the Auckland Medical Research Foundation and The University of Auckland Doctoral Scholarship to WJT Tan.

Effects of cochlear implant use on binaural processing

James B. Fallon, Sam Irving, Andrew K. Wise, Robert K. Shepherd, Dexter R.F. Irvine

Bionics Institute and the , , Australia

Introduction: Bilateral cochlear implantation is increasingly common, particularly for young children, and results in an increase in performance for both sound localization and speech discrimination in noise compared to unilateral implantation. However, the improvements are small and performance remains inferior to that of normal listeners. Animal and psychophysical studies have shown that long-term deafness from a young age degrades processing of interaural time differences (ITDs) but not interaural level differences (ILDs). The effects of chronic bilateral cochlear implant use on binaural processing are less clear.

Methods: Three groups of cats were used: two normal hearing controls (NHC), two neonatally profoundly deafened unstimulated cats (NDUS) and four neonatally profoundly deafened cats that received approximately 6 months of bilateral intra-cochlear electrical stimulation from clinical cochlear implants and speech processors (NDS). Single-unit responses (n= 110, 60, 86 for the NHC, NDUS and NDS groups respectively) were bilaterally recorded from the central nucleus of the inferior colliculus in response to electric binaural stimulation with a range of ITDs and ILDs using 32 channel silicon arrays (NeuroNexus).

Results: ITD sensitivity was significantly (Kruskal-Wallis test, p < 0.05) poorer in both the neonatally deafened groups compared to the normal hearing animals, and there was no difference between the stimulated and unstimulated groups (p > 0.05). ILD sensitivity was not different between the groups (p > 0.05).

Conclusions: The use of bilateral clinical cochlear implants does not prevent / reverse the degradation in ITD processing resulting from long-term deafness from a young age. Whether experience with appropriate ITD cues would improve ITD processing still needs to be examined. ILD processing is largely unaffected by both long-term deafness and chronic stimulation.

This work was funded by the NH&MRC and The Bionics Institute acknowledges the support it receives from the Victorian Government through its Operational Infrastructure Support Program.

We thank Nicole Critch, Amy Morley, Damian Robb for technical assistance.

HAIR CELL REGENERATION BY ATOH1 GENE THERAPY IN THE COCHLEA OF MATURE DEAFENED GUINEA PIGS Rachael T Richardson1,2,3, Patrick J Atkinson1,2, Brianna O Flynn1, Bryony A Nayagam1,2,4 and Andrew K Wise1,2,3

1Bionics Institute, Melbourne, Australia 2University of Melbourne, Department of Otolaryngology, Melbourne, Australia 3University of Melbourne, Department of Medical Bionics, Melbourne, Australia 4University of Melbourne, Department of Audiology and Speech Pathology, Melbourne, Australia

Background and Objectives: Degeneration of hair cells in the mammalian cochlea results in irreversible hearing loss. Cochlear implants restore hearing to people with profound hearing loss, but have limitations such as the inability to convey music. Hair cell regeneration may provide a more natural hearing solution. The Atoh1 gene is necessary for hair cell development and recent research has shown that Atoh1 gene therapy results in new hair cell formation and hearing restoration. The aim of this study was to characterise hair cell formation via Atoh1 gene therapy in noise-deafened mature guinea pigs. Methods: Guinea pigs were deafened by noise (130 dB, 11-13 kHz, 2 hours). After 2 weeks, the left cochleae were injected with an adenoviral vector containing the Atoh1 gene with a GFP reporter gene (Atoh1 group). Control animals were injected with a GFP-only control adenoviral vector (GFP group). Two-week deaf–only and 5-week deaf-only control groups were also included in the study. Three weeks after injection cochleae were assessed for hair cell number, maturity and hair cell synaptogenesis with auditory neurons. Hearing thresholds were assessed throughout. Results: There were significantly more myosinVIIa+ hair cells in cochleae over-expressing Atoh1 compared to the contralateral cochlea and compared to control guinea pigs (p<0.05 one way ANOVA), however, the number of hair cells in Atoh1-treated animals was far below normal. Expression of Atoh1 had a positive impact on the preservation of the cytoarchitecture of the sensory epithelium compared to controls (p<0.001 one way ANOVA). Expression of the synaptic protein CtBP2 was present in some GFP+ Atoh1 cells but was reduced in density. There was some evidence of recruitment of auditory neurons towards GFP+ Atoh1 hair cells (p<0.05 one way ANOVA), but hearing was not restored. Conclusions: Atoh1 gene therapy alone cannot fully convert non-sensory cells into new hair cells after noise-induced hearing loss. This study revealed that the degree of degeneration at the time of gene therapy has a big impact on whether Atoh1 gene therapy can yield functional hearing improvements.

Key words: Atoh1; Gene therapy; Hair cells; Noise; Regeneration

Acknowledgements: This study was generously funded by Action on Hearing Loss, the Garnett Passe and Rodney Williams Memorial Foundation and the National Health and Medical Research Council (GNT1024350). The Bionics Institute acknowledges the support it receives from the Victorian Government through its Operational Infrastructure Support Program

CHD7 Deficiency in “Looper”, a New Mouse Model of CHARGE Syndrome, Results in Ossicle Malformation, Otosclerosis and Hearing Impairment Jacqueline Ogier Molecular Hearing Laboratory, Murdoch Childrens Research Institute, Parkville 3052, Australia

CHARGE syndrome is a rare disorder that affects 1/10,000 live births and is caused by mutations in the gene encoding chromodomain helicase DNA binding protein 7 (CHD7). This is a complex syndrome with clinical features including, growth retardation, facial asymmetry, vestibular defects, eye anomalies, hyperactivity, ossicle malformation, hearing loss and vestibular dysfunction. The function of CHD7, and the mechanism by which CHD7 deficiency affects this diverse range of body systems during development, remains unclear. During a genome-wide mouse mutagenesis screen for new models of deafness that our laboratory conducted, a mouse with CHARGE-like characteristics was detected. The mutant strain was named “Looper” in reference to its characteristic circling behavior. Meiotic mapping and exome sequencing of this strain revealed a novel autosomal dominant mutation (c.5690C.A, p.S1897X) in the Chd7 gene. Subsequent phenotypic assessment of the strain using a battery of behavioural, histological and electrophysiological techniques demonstrated numerous similarities between Looper pathology and that of human CHARGE syndrome. In particular, Looper mice have an otosclerosis like fusion of the stapes footplate to the cochlear oval window leading to hearing impairment. In addition, Looper mice were found to be hyperactive and had severe vestibular dysfunction but surprisingly did not display motor impairment. Furthermore, blepharoconjunctivitis but not coloboma was observed in this strain. These results indicate that the Looper mouse is an excellent model of CHARGE syndrome that will be useful for further investigation of the disease and CHD7 specific developmental pathways.

Circuitry between the Dorsal Cochlear Nucleus and the Inferior Colliculus in the mouse

Giedre Milinkeviciute1,2, Michael A. Muniak1, Tan Pongstaporn1, Annie Cho1, David K. Ryugo1,2 1Garvan Institute, Darlinghurst, NSW, 2School of Medical Sciences, University of , Sydney, NSW

Background

The central auditory system is comprised of numerous auditory nuclei that are connected by ascending and descending projections. The cochlear nucleus (CN) and inferior colliculus (IC) are two major structures with complementary interconnections, much of whose detailed nature remains to be elaborated. In order to infer mechanisms of signal processing at this early stage, it is essential to establish how these structures are connected. This project is focused on the synaptic connections between the dorsal cochlear nucleus (DCN) and the IC.

Methods

A cocktail of anterograde and retrograde tracer dyes was injected into the DCN of CBA/CaH mice in the awake animal after determining multiunit best frequency and threshold. Brains were processed using standard histologic methods for visualization and examination of neuronal tracer dyes using fluorescence, bright field, and electron microscopy (EM), and axons and cells were traced.

Results

Some if not all of the ascending DCN fibres that terminate in the ipsilateral IC reach this location by traveling through the contralateral IC and crossing over via the commissure. Terminal fields were observed in the dorsal (DCIC) and external cortices (ECIC) of the IC. Preliminary data suggest reciprocal contralateral connections between the IC and the DCN that establish feedback and feedforward loops. EM analysis seeks to confirm synapses between labelled endings and retrogradely filled cells in the IC. There are numerous retrogradely filled cells in the ipsilateral IC. The ratio between labelled cells in the ipsilateral and contralateral sides is 3:1, respectively. Although the majority of labelled cells seem to be contained within the central nucleus of the IC (CNIC) bilaterally, cells in the ipsilateral side are found in other IC subdivisions as well. It is yet to be determined whether cells in the CNIC are contained within the same frequency band.

Conclusions

The multiple pathways between the DCN and the IC emphasize considerable complexity in how auditory signals are processed. A frequency-specific enhancement of acoustic signals is implied through the reciprocal contralateral circuit. Feedback from the ipsilateral IC might strengthen the signal through additional reverberant excitation, lateral inhibition, or even both. These findings would implicate a role for the DCN-IC circuit in signal enhancement in noisy backgrounds. Moreover, ascending DCN terminals in the DCIC and ECIC might be synapsing onto the cells that receive descending input from the auditory cortex, creating a circuit for cortical modulation of sound processing through links to memory and emotion.

FOCUSED MULTIPOLAR STIMULATION FOR IMPROVED PERFORMANCE OF COCHLEAR IMPLANTS: PRECLINICAL STUDIES

SS George1,2, RK Shepherd1,2, AK Wise1,2, MN Shivdasani1,2 and JB Fallon1,2

1Bionics Institute, East Melbourne, Victoria. 2Department of Medical Bionics, The University of Melbourne, Parkville, Victoria.

Aim: The research evaluated the efficacy of a current-focusing technique – focused multipolar (FMP) stimulation – in producing focused activation in the cochlea by measuring neural activation in the auditory midbrain.

Background: The conductive fluids and tissues of the cochlea lead to the spread of electrical fields and broad neural excitation using contemporary cochlear implant stimulation configuration such as monopolar (MP) stimulation. Focusing of the stimulation is expected to result in better performance.

Methods: Following implantation of Cochlear™ Hybrid-L 14 array into the acutely (n=8) and long- term deafened (n=8) cochlea of cats, the inferior colliculus (IC) contralateral to the implanted cochlea was exposed. Multiunit responses were recorded across the cochleotopic gradient of the central nucleus of the IC in response to electric (MP and FMP) stimulation over a range of intensities, using a 32 channel silicon array (NeuroNexus). The spread of activation was quantified using spatial tuning curves (STCs) across the IC.

Results: MP resulted in significantly wider STCs compared to FMP in both groups (one-way RM ANOVA, p<0.001). However, thresholds were significantly higher (one-way RM ANOVA, p<0.001) for FMP compared to MP. No significant difference in the STC width was observed (t-test, p>0.05) for the different electrode array positioning.

Conclusions: The data suggest that FMP results in more restricted neural activation than MP and this advantage of FMP is maintained in cochleae with significant neural degeneration that reflects the clinical situation. The greater spatial selectivity of FMP would be expected to result in improved clinical performance.

Funding: This work is funded by the Garnett Passe, Australian Postgraduate Award (APA) and Bart Reardon Scholarship. The Bionics Institute acknowledges the support it receives from the Victorian Government through its Operational Infrastructure Support Program.

Effect of Asymmetrical Hearing Loss in the Inferior Colliculus

1,2Basil Razi, 1Michael A. Muniak, 1,2David K. Ryugo 1Garvan Institute of Medical Research, Sydney, Australia 2University of New South Wales, Sydney, Australia

Background: Approximately 20% of Australians suffer from hearing loss (HL), with an increase to 25% projected by 2050, with asymmetrical HL being much more common than a bilateral symmetrical loss. HL is devastating to normal socialisation and when lost at a young age, places the individual at a significant disadvantage in the educational system. Current treatment for asymmetrical HL involves cochlear implants and/or hearing aids depending on the extent of loss. Outcomes, however, are variable and less successful for profoundly deaf children. Recent data suggest that poor treatment outcomes may be associated with degraded underlying brain processes caused by age of onset, duration, and degree of deafness. One process that is degraded with HL is frequency discrimination, which in the normal brain, is achieved through the layout and interplay of neurons that maintain a frequency specific organisation in the central auditory system. In order to understand how the organisation of the central auditory system might change when hearing is lost, we sought to investigate the effects of conductive and sensorineural asymmetrical HL on the fibro-dendritic organisation within the inferior colliculus (IC) of mice with different ages of HL onset.

Methods: Mice were surgically deafened at P10 or P60 by either ossicular removal (conductive HL) or drainage of cochlear endolymph (sensorineural HL). Following a 28-day survival, IC cells and their dendrites were visualised using Golgi-Cox staining. Analysis of orientation of dendrites was carried out using Neurolucida and MatLab programs on reconstructed, Golgi-stained neurons.

Results: Dendritic orientation of neurons in the IC in both conductive HL and surgically deafened mice deviate from the bidirectional laminar organisation seen in sham operated controls. Moreover, mice with sensorineural deafness exhibit shorter and more disarrayed dendrites than conductive HL mice. No strong relationship was seen between tested age of HL onset and dendritic organisation in the IC.

Conclusions: This study demonstrates that asymmetric HL results in a disruption of the dendritic organisation in the IC, with a greater detrimental effect arising from sensorineural deafness. Our findings suggest that structural changes of the auditory midbrain involve a disarray of the fibro-dendritic organisation in the IC. This aberration likely plays a crucial role in the functional outcomes of HL treatment. Future studies will be needed to describe in detail the effects of asymmetric HL on the incoming axonal organisation, and whether cochlear implants and/or hearing aids can rescue the structural disorganisation. Deafness, neurotrophins and cochlear implants

R. Shepherd1,2, A. Wise1,2, R. Pujol1,3, R. Richardson1,2, L. Gillespie1,2 and J. Fallon1,2 1Bionics Institute; 2University of Melbourne, Melbourne, Australia and 3 Inserm U 583, University Montpellier, Montpellier, France

While neural prostheses provide significant improvements in clinical outcomes for a variety of sensory and neurological disorders, many of these devices stimulate damaged neurons either as a result of the underlying aetiology or due to trauma associated with the implantation of the electrode array. We believe there is an opportunity to improve the long-term status of the electrode-tissue interface by combining neural prostheses with therapeutic drug delivery, particularly during the surgical implantation of the device. Over the last decade we have examined a number of drug delivery techniques in animal models of cochlear implants; this abstract summarises aspects of this research.

Our work has focussed on the delivery of exogenous neurotrophins to promote the rescue of auditory neurons following deafness. While drug-delivery via pumps is an efficient procedure for relatively short periods of time, the use of a canula to deliver the drug to the cochlea and the ongoing need to replenish/replace the pump results in a risk of infection. We have developed alternative delivery methods for long term (weeks-months) drug delivery including the use of viral vectors, cell-based therapies and slow-release nanotechnology-inspired applications. All these techniques can be implemented in association with cochlear implant surgery; moreover our functional studies have demonstrated that chronic neurotrophin delivery results in reduced electrical thresholds.

Importantly, a number of the technologies described have the potential to deliver a wide variety of therapeutic drugs in a highly controlled manner. We consider that long-term drug delivery in concert with neural prostheses has great potential to improve the electrode-neural interface.

This work was supported by the NIDCD (HHS-N-263-2007-00053-C), the Garnett Passe and Rodney Williams Memorial Foundation, Action on Hearing Loss, the Victorian Government and the National Health and Medical Research Council of Australia.

The Source of Infrasonic Cochlear Microphonic Potentials in the Guinea Pig Cochlea Dominic Lopez University of Sydney

Background: How the Endolymphatic Potential (EP) and inner ear fluid volumes are regulated is presently unclear, but is likely to involve some form of voltage and/or stretch receptor. A recent study by Salt et al., (2013) demonstrated that very low-frequency (LF; <20Hz) tones evoked abnormally large Cochlear Microphonic (CM) potentials, which could not be explained on the basis of outer hair cell mechano-electrical transduction (MET) channels alone. Methods: Small holes were made in the bony lateral of the cochlea in anesthetised guinea pigs to allow the insertion of glass micropipettes into Scala Media (SM), Scala Tympani (ST), Scala Vestibuli (SV) or in the basal wall of Stria Vascularis. Distorted CMs evoked by continuous tones between 4 Hz to 500 Hz were recorded under different physiological conditions, including the simultaneous presentation of HF and LF tones, or during acute asphyxia. The output of a first-order Boltzmann function was mathematically fitted to the CM waveforms, providing characteristics of the CM, such as the DC offset (Voff), the saturating voltage (Vsat), and the asymmetry (OP). The electrical impedance of the cochlear compartments was measured by injecting AC current into SM at different frequencies and simultaneously measuring the voltage fluctuations in SM. Results: The saturated LF CM recorded in SM was almost twice as large at the saturated HF CM. Saturated CMs measured in SV and ST were all of near-equal amplitude. When the cochlea was exposed to a LF and HF tone simultaneously, the resultant CM measured in SM suggested that the HF CM was ‘riding’ upon a large cyclic voltage fluctuation that was not generated by the MET channels alone. Similar results were not observed in ST and SV recordings. In acute hypoxic conditions the large Vsat for LF CMs disappeared, but returned upon recovery. The injection of AC current in to SM suggested that the cochlear tissues are simply resistive below 500 Hz. Conclusions: If the CM was generated entirely as an ohmic drop across the cochlear partition, we would anticipate that the CM should simply invert polarity between SM and ST, and that the frequency response of the CM would be similar in SM and ST. This was not the case - begging the question - where does the additional voltage generating the large LF CM measured in SM come from? Electrical impedance measures suggest the voltage changes are not related to the impedance of the cochlear tissues, and hypoxia experiments suggested that the large LF voltage component was dependent upon normal function of Stria Vascularis. We are currently performing additional experiments to determine the origin of this large LF CM phenomenon. Given that the enhanced Vsat only occurs at infrasonic frequencies, it is possible that this additional LF CM generator is involved in the homeostatic regulation of EP and cochlear fluid volumes.

Behavioural and Neural Correlates in an Animal Model of Tinnitus & the Therapeutic Potential of Acute Electrical Stimulation of the Round Window

Emma Johnson1,2

1Bionics Institute, East Melbourne, Victoria. 2Department of Medical Bionics, The University of Melbourne, Parkville, Victoria.

ABSTRACT The subjective nature of tinnitus has limited past research on underlying neural mechanisms and potential therapies. Behavioural measures of tinnitus in animal models have allowed reproducible studies and have begun to shed light on neural correlates. The current study sought to produce a behavioural animal model of tinnitus from noise induced hearing loss that could be correlated with neural and histological markers. Of particular interest to the Bionics Institute was a preclinical model that could test the therapeutic potential of bionic devices. The current study examined the acute effects of a Round Window Implant, as a less invasive alternative to a Cochlear Implant, on spontaneous firing rates.

Guinea pigs’ ABR threshold losses were measured immediately and 2 weeks following unilateral noise exposure to a 10 kHz tone. A ‘Gap Startle Test’ was used to manipulate the Prepulse Inhibition of the Acoustic Startle Reflex to test for tinnitus that matched 8, 10, 12 or 14 kHz background noises following noise deafening. Spontaneous firing rates were measured across the tonotopic arrangement of the Inferior Colliculus 2 weeks post noise deafening, before and after 1 hour of electrical stimulation by a Round Window Implant. Immunohistochemistry was used to examine the basal turn of the noise exposed ear for inner and outer hair cell lesions.

A temporary threshold shift and permanent threshold shift was observed following noise deafening. Behavioural evidence of tinnitus was observed in half the noise deafened animals. Inner and outer hair cell lesions were found. No significant differences between spontaneous firing rates of single units in noise deafened regions and outside noise deafened regions in the Inferior Colliculus were found. Following Round Window electrical stimulation, unexpected significant increases in the spontaneous firing rates of single units outside noise deafened regions were observed. Putative Contacts of Medial Olivocochlear Collaterals in the Ventral Cochlear Nucleus of the Rat Ahmaed Baashar, Donald Robertson and Wilhelmina Mulders The Auditory Laboratory, School of Anatomy, Physiology and Human Biology, University of , Perth, Australia

The auditory system is composed of ascending and descending auditory pathways. The latter play an important role enabling the brain to modify afferent auditory input before it reaches the cortex. An important component of the descending pathways is the olivocochlear system which originates in the brainstem and projects out to the cochlea. A subpopulation of olivocochlear system, medial olivocochlear (MOC) neurons, gives off collateral branches to the cochlear nucleus which have been shown to make synaptic contacts with dendrites of multipolar neurons in the ventral cochlear nucleus. These multipolar neurons, however, consist of two distinct neuronal populations: T-stellate neurons, thought to project to the inferior colliculus and D-stellate neurons, thought to project to the contralateral cochlear nucleus. Each of these neuronal subtypes serve a variety of roles in auditory processing and it is therefore essential to determine which of them is innervated by the olivocochlear collaterals in order to shed light on the possible role of the olivocochlear collaterals in auditory processing. At present this is still unclear since a conflict exists between results obtained from in vivo electrophysiological studies (Mulders et al., 2003, 2007, 2009) and in vitro pharmacological studies (Fujino and oertel, 2001). This project aims to identify which neural cell types in the cochlear nucleus receive synaptic innervation from the MOC collaterals using anatomical techniques. The retrograde tracer Fluorogold was injected into the inferior colliculus or cochlear nucleus to label T-stellate and D-stellate neurons in the ventral cochlear nucleus, respectively. Axonal branches of MOC neurons were labelled by injections of biocytin at the floor of the IVth ventricle. Fluorogold injections into the inferior colliculus or cochlear nucleus both resulted in labelled multipolar neurons in the cochlear nucleus. Biocytin labelling resulted in labelled MOC collaterals in the same areas as described previously. Microscopic analysis revealed that MOC collaterals make some putative synaptic contacts with the cell bodies and dendrites of the neurons labelled following injections of the inferior colliculus and cochlear nucleus. Our results suggest that both populations of multipolar neurons in the ventral cochlear nucleus receive synaptic innervation from the collaterals of MOC neurons.

FUJINO, K. & OERTEL, D. 2001. Cholinergic modulation of stellate cells in the mammalian ventral cochlear nucleus. The Journal of Neuroscience, 21, 7372-7383.

MULDERS, W. H. & ROBERTSON, D. 2009. Hyperactivity in the auditory midbrain after acoustic trauma: dependence on cochlear activity. Neuroscience, 164, 733-746.

MULDERS, W. H. A. M., HARVEY, A. R. & ROBERTSON, D. 2007. Electrically evoked responses in onset chopper neurons in guinea pig cochlear nucleus. Journal of neurophysiology, 97, 3288-3297.

MULDERS, W. H. A. M., PAOLINI, A., NEEDHAM, K. & ROBERTSON, D. 2003. Olivocochlear collaterals evoke excitatory effects in onset neurones of the rat cochlear nucleus. Hearing Research, 176, 113-121. Brain Changes in the Cochlear Nucleus that Accompany Hearing Loss 1Catherine Connelly, 2Amanda Lauer, 1Kirupa Suthakar, 1Tan Pongstaporn, 1Annie Cho, 1,2David K. Ryugo 1Garvan Institute of Medical Research, Sydney, NSW, Australia 2Johns Hopkins University, Baltimore, MD, USA

Background: Synaptic features of the central nervous system change as a consequence of alterations in sensory input. Studies of partially deaf cats show atrophic changes in brainstem synaptic morphology and postsynaptic ultrastructure (Ryugo et al., 1998), and congenitally deaf white cats exhibit even more severe brainstem pathologies, such as synaptic hypertrophy (Ryugo et al., 1997) and a redistribution of excitatory and inhibitory synaptic terminals (Tirko et al., 2009). This study aims to identify the sequelae of hearing loss in the central auditory system of mice with hearing loss.

Methods: Three mouse strains (aged 1, 3, and 6 months) representing distinct hearing phenotypes were used: CBA/CaHs with normal hearing thresholds through adulthood; DBA/2s, exhibiting progressive high frequency hearing loss starting at one month of age; and homozygote congenitally deaf shaker-2s. Cochlear nuclei from mice of various ages and degrees of hearing loss were immunostained using anti-GlyT2 and anti-ChAT antibodies. The organization of inputs to bushy cells were analyzed with light and electron microscopy.

Results: Synapses of auditory nerve fibers to bushy cells in CBA/CaH hearing mice are dome- shaped with prominent PSDs, synapses of deaf shaker-2 mice are elongated and flattened, while synapses of the DBA/2 exhibit synapse morphology that appears graded between the two. With respect to non-primary inputs to the bushy cell, the amount of glycinergic and cholinergic terminals remains constant from 1 to 6 months of age in the CBA/CaH mouse. However, the deaf shaker-2 and DBA mice have a reduction in the amount of glycinergic terminals around their bushy cells, whereas the number of cholinergic terminals increases. The amount of change is related to hearing status.

Conclusion: Preliminary data show that congenital and acquired hearing loss result in central auditory pathologies. Loss of inhibitory terminals and proliferation of cholinergic terminals suggest a release of inhibition and expansion of excitation, respectively. These changes may give insight to mechanism that underpin tinnitus. Based on these results, it becomes prudent to determine which abnormalities can be remedied by treatment methods such as sound amplification. Future studies will test the hypothesis that an amplified sound environment prevents or delays the progression of brain pathologies in comparison to that of long-term, unattended hearing loss.

Effects of Sound Amplification After Hearing Loss on Central Auditory Plasticity

Femi Ayeni1, 2, Michael Muniak1, Radha Simhadri3, David Ryugo1, 2. (1) Hearing Research, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia; (2) School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia; (3) Hearing, Tinnitus & Implant Centre, Wollongong, NSW, Australia. [email protected] Introduction Sensory deprivation and/or stimulation modulates the central nervous system through plastic changes. Diminished auditory input caused by hearing loss can disturb the functional and anatomical integrity of structures involved in auditory processing. Peripheral hearing loss is typically treated with hearing aids that amplify acoustic signals, but it is unknown if their use can also mitigate and/or restore changes to central pathways. To address this question, we simulated hearing aid use in DBA/2 mice—a model of progressive peripheral hearing loss—by providing acoustic stimulation specific to the hearing loss profile of each animal. We subsequently examined auditory brain stem responses (ABRs) and the morphology of auditory nerve endings onto bushy cells (endbulb of Held) in the cochlear nucleus.

Method DBA/2 mice were housed in customised acoustic chambers fitted with overhead speakers. Audiograms were collected weekly from three weeks of age for both experimental and control mice. Animals began stimulation at varying age points, receiving baseline stimulation at 55 dB SPL (RMS). Experimental animals received additional frequency-specific amplification based on the hearing loss profile of weekly audiograms. Stimulation was provided 12 hours nightly for eight weeks and consisted of environmental and synthesized sounds spanning the full hearing range randomly interleaved with equal amounts of silence.

After the study period, each animal received an injection of an anterograde tracer (neurobiotin) into the auditory nerve and was allowed to survive 4-6hrs. Brains were processed to reveal labeled endbulbs of Held using a fluorescent marker. Image stacks were acquired with a confocal microscope (Leica DMI 6000 SP8) using fixed image acquisition parameters. Endbulb stacks were ported to Imaris software (Bitplane) for manual thresholding and analysis. The same median filter was applied to all images and the observer was blinded from image acquisition through data analysis.

Results ABR audiograms suggest that central auditory structures in mice receiving stimulation become more excitable as evidenced by improved response latency and late ABR wave morphology at mid- frequencies (16-32 kHz @ 90dB). Anatomical analyses further suggest that endbulb morphology may be better preserved in mice that commence stimulation at earlier ages.

Conclusion Our data suggest that the benefits of hearing aids may help to prevent or mitigate synaptic abnormalities in the central auditory system induced by hearing loss. This effect appears to be more robust if stimulation is provided early, providing an argument for early adoption of hearing aids when hearing loss is detected.

Culture conditions affecting spiral ganglion neuron physiology Helen Cai University of Melbourne Neurotrophins (NTs) and antibiotics are commonly used as supplements in cell cultures. Cells are highly susceptible to changes in the extracellular environment and require time to adjust to the culture environment. Both NTs and antibiotics can alter ion channel expression and activation, thus affecting the firing properties of cells. In addition, incubation time may also affect cell proper- ties as cultured cells overcome the initial dissociation trauma. This may affect their in vitro physi- ology when assessing the influence of exogenous agents.

This study investigated the effect of time, NTs (brain-derived neurotrophic factor and neurotro- phin-3) and antibiotics (penicillin and streptomycin) on spiral ganglion neurons (SGNs) in vitro. Whole-cell patch clamp recordings were conducted on 182 SGNs at 1 and 3 days in vitro (DIV) to analyse the effects of NT and antibiotic treatment on SGN firing profiles. Immunocytochemistry was used to assess transient receptor potential vanilloid-1 (TRPV1) expression in 153 SGNs and activating transcription factor-3 (ATF3) expression in 146 SGNs at 4 hours, 1 DIV, 2 DIV and 3 DIV. Immunolabelling was compared across time and between the different NT and antibiotic treat- ments.

There was an increase in action potential latency and a decrease in threshold over time regardless of treatment. No slowly adapting SGNs that fire more than six APs were present at 1 DIV. The ef- fects of NT were additionally affected by the recording day. Antibiotics affected more firing prop- erties of 1 DIV SGNs compared to 3 DIV SGNs. TRPV1 expression was initially upregulated and de- creased over time while ATF3 expression increased over time. The findings of this study suggest 1 DIV SGNs are more sensitive to the culture environment. As such 3 DIV would be more suitable for future in vitro studies compared to 1 DIV.

Effect of Nucleus Accumbens Stimulation on Medial Geniculate Nucleus Neurons

Kristin M. Barry, D. Robertson, A.G. Paolini, W.H.A.M. Mulders

Affiliations: The Auditory Laboratory, School of Anatomy, Physiology and Human Biology, University of Western Australia

Tinnitus is a phantom perception of sound known to be associated with noise-induced hearing loss (NIHL). Many tinnitus sufferers experience debilitating distress, depression and insomnia and there is currently no cure. In animal models of NIHL, it has been demonstrated that abnormal neural activity develops in the auditory pathway and this has been suggested to be involved in tinnitus generation. However, this abnormal neural activity does not always lead to the development of a tinnitus percept leading researchers to consider other brain regions which may be involved in tinnitus generation.

Recently, evidence has emerged for the involvement of limbic structures in the generation of tinnitus. Limbic circuitry is proposed to distinguish between salient and abnormal neural activity at the level of the medial geniculate nucleus (MGN) of the thalamus (Rauschecker et al. 2010). In tinnitus, a failure of this circuitry may result in abnormal neural activity being brought to conscious perception. Interestingly, in humans with tinnitus, structural changes and abnormal activity have been found to occur in parts of the limbic system such as the nucleus accumbens (NAc) (Mühlau et al. 2006). However, at present, no studies have been conducted on the influence of the NAc on the MGN.

We investigated the functional connectivity of the NAc on MGN single neurons. Anesthetized Wistar rats were placed in a dual manipulator stereotaxic apparatus. A bipolar stimulation electrode was positioned in the NAc while a recording electrode was placed in the MGN. Using single shocks (0.5 ms pulse duration; range of 0-1mA), effects of NAc shocks on spontaneous firing rates of MGN neurons were recorded. Electrical stimulation of NAc did result in a 20ms period of suppressed MGN spontaneous firing rates in some but not all MGN neurons recorded. The result provides support for the existence of circuitry by which NAc activation could modulate the activity of auditory neurons in the MGN.

References:

Mühlau, M. et al., 2006. Structural brain changes in tinnitus. Cerebral cortex (New York, N.Y. : 1991), 16(9), pp.1283–8. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16280464 [Accessed September 18, 2013].

Rauschecker, J.P., Leaver, A.M. & Mühlau, M., 2010. Tuning out the noise: limbic-auditory interactions in tinnitus. Neuron, 66(6), pp.819–26. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2904345&tool=pmcentrez &rendertype=abstract [Accessed September 17, 2013].

Inferior Collicular Input to Olivocochlear Efferents in Hearing and Hearing Loss

1,2Kirupa Suthakar, 1Hannah Douglas-Kinninburgh, 1Tan Pongstaporn, 1Annie Cho, 1,2David Ryugo 1Garvan Institute of Medical Research, Sydney, NSW, Australia 2UNSW Australia, Sydney, NSW, Australia

Background The descending auditory system, which parallels the ascending pathways, consists of long projections and shorter neuronal chains that modulate the incoming acoustic signals. The olivocochlear (OC) efferent system is the end point of the descending system receiving both ascending and descending input, which allows the brain direct control of the peripheral auditory system. Descending topographic projections from the inferior colliculus (IC) to the location of these OC efferent cell bodies support the idea of excitatory activation of OC efferents (Vetter et al., 1993; Mulders & Robertson, 2000&2002; Zhang & Dolan, 2006). Two proposed functions of the OC system are protection from acoustic trauma and modulation of sensory input. This study aims to investigate the anatomical nature of descending IC input onto OC efferents in mice with normal hearing, hearing loss, and congenital deafness in order to better understand why impaired speech understanding accompanies hearing loss.

Methods Adult, hearing CBA/Ca mice received small injections of biotinylated dextran-amine (BDA) into frequency-specific regions of the central nucleus of the IC to study labelled terminals in the SOC, and in some cases also received CTB injections in the cochlea to visualise OC efferent cell bodies. The connections of normal hearing CBA/Ca mice were compared to that of ‘early onset hearing loss’ (DBA/2) and ‘congenital deafness’ (Shaker2). Analysis was directed to identify direct synaptic contact onto OC efferent cell bodies using light and electron microscopy.

Results Preliminary data confirm that descending IC input is arranged tonotopically. In the normal hearing condition, terminal boutons from lower frequency IC injections appear more laterally located in SOC nuclei. Qualitatively, the terminal bouton fields are all located more laterally in each of the groups; however in the congenitally deaf animals the terminal bouton field appears truncated and condensed. Moreover, labelled IC terminal boutons make direct synaptic contact with labelled OC efferent cell bodies. These labelled terminals contain round synaptic vesicles and asymmetric synapses, indicative of excitatory neurotransmission.

Conclusion Our results indicate that the IC provides excitatory tonotopic input to OC efferents. The IC is ideally situated to provide multi-modal integration for complex filtering tasks at the earliest stages of central auditory processing. Preliminary data suggest that pathological hearing situations may result in reorganisation of descending inputs from the IC to OC efferents. Alterations in the descending auditory projections may contribute to impairment of speech discrimination in noise, which is a common symptom of hearing loss. The effects of combined neurotrophin treatment on spiral ganglion neuron electrophysiological properties and outward potassium currents Tess Wright1, Lisa Gillespie2,3, Karina Needham1 1 Department of Otolaryngology, University of Melbourne; 2 Bionics Institute; 3 Medical Bionics Department, University of Melbourne

Neurotrophin (NT) therapy is a promising adjuvant to improve the efficacy of the cochlear implant. NT therapy has been shown to support and protect spiral ganglion neurons (SGNs) in vivo and in vitro. Neurotrophic supplements can, however, also modify firing properties, ion channel expression and ion channel activity in SGNs. We aimed to investigate what NT mediated effects are happening in vitro, particularly to the potassium currents and basic electrophysiological properties of SGNs.

Whole-cell patch clamp recordings were performed at two days in vitro (2DIV) on SGNs that had no NT treatment and on SGNs that had combined treatment with brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3). Voltage and current clamp were used to analyse firing properties and outward potassium currents (IK) of treated and untreated SGNs. Contaminating + currents; Na and Ih currents were removed respectively by 4-ethylphenylamino-1,2-dimenthyl-6 methylaminopyrimidinium (ZD7288) and Tetrodotoxin (TTX). In order to isolate potassium currents of interest; low voltage activated current (IKL) and A-type (IA), dentroxin-I (DTX-I), alpha- dendrotoxin (α-DTX) and phrixotoxin (PaTX-1) were utilised to block specific subunits.

Analysis of firing properties revealed that NT-treated SGNs had prolonged latencies in comparison to control SGNs. Interestingly, all other basic firing properties were not affected by NT treatment.

Voltage clamp revealed that NTs had no effect on IK. The blocking of KV1.1 and KV1.2 (by DTX-I) inhibited the IKL and revealed no differences in the reduction of current between experimental groups. The IA was blocked by inhibiting KV4.2 and KV4.3 (by PaTX-1) and also showed no difference between groups. However, the removal of the IKL current by α-DTX (KV1.1, KV1.2 and

KV1.6 blocker) demonstrated a significantly larger reduction in control SGNs than NT-treated SGNs at -3mV, indicating that the KV1.6 subunit contributes significantly in control SGNs.

These results suggest that NT-treatment is affecting SGN firing properties and that the α-DTX- sensitive current is contributing greater to the IK in control SGNs. These changes to SGN properties in vitro may also be influenced in NT treatment in vivo and could influence the effectiveness of NT therapy.

Human vestibular hair cells develop earlier than cochlea hair cells Lim R, Drury HR, Camp AJ, Tadros MA, Callister RJ, & Brichta AM.

Introduction The majority of studies investigating the development of peripheral vestibular function have focused on animal models. Here we describe anatomical and physiological characteristics of developing human inner ear hair cells during a period of maturation between 12 and 18 weeks gestation (WG).

Methods Tissue was electively donated and used for anatomical or electrophysiological studies. For anatomical studies, human inner ear tissue was fixed using 4% paraformaldehyde, and then sectioned. A number of different antibodies were used to label hair cells, stereocilia, and calcium binding proteins. For electrophysiological studies, the vestibular triad including semicircular canal cristae and utricle were excised in ice-cold glycerol-based Ringers’ solution then transferred to a recording chamber perfused with oxygenated L15 cell culture medium. Whole-cell patch-clamp recordings using potassium fluoride internal solution were made from embedded hair cells.

Results Our anatomical results show there is a diversity of morphological characteristics (cylindrical versus amphora shape) of developing human vestibular hair cells by 13 WG. In vestibular organs, we also have evidence for polarity of hair bundles with the expression of acetylated tubulin by the kinocilium as distinct from phalloidin expression by stereocilia by 13 WG. In contrast, however, at 13WG, the various cell types had only just begun to differentiate in the cochlea. We have recorded and intracellularly labelled human vestibular hair cells that display inward and outward rectifying conductances. Throughout the period examined (11-18 WG), approximately 20% of immature hair cells exhibit sodium conductances. Cells classified as type II hair cells, due to lack of Gk,l conductance, showed a significant increase in Gmax between 11-14 WG and 15-18 WG (3.5 ± 0.2 nS versus 11.9 ± 1.5 nS, p<0.05). The earliest expression of the mature type I hair cell conductance, Gk,l was observed at 15 WG. This approximately coincides with our first recordings from calyx afferent terminals (15 WG). Due to later maturation of cochlea hair cells no recordings were obtained

Conclusion Our data show that while human vestibular hair cells are beginning to show distinct morphological changes by 13 WG,functionally hair cells aged b/w 11- 14 WG still express immature conductances. By 15 WG, vestibular hair cells begin to express more mature conductances, including those typically seen either in mature type I or type II hair cells. There is a concomitant maturation of calyceal afferent terminals contacting putative type I hair cells. Cochlea hair cells were too immature for recording purposes.