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Resting neural activity patterns in auditory brain areas following conductive hearing loss by

Jaina Negandhi

A thesis submitted in conformity with the requirements for the degree of Master in Science Graduate Department of Physiology University of Toronto

© Copyright by Jaina Negandhi 2012

Resting neural activity patterns in auditory brain areas following conductive hearing loss Master of Science, 2012 Jaina Negandhi Department of Physiology, University of Toronto

Abstract Introduction: A common cause of conductive hearing loss in young children is otitis media, which when chronic or recurrent can effect speech and language development. However, little is known about the effects of such conductive loss on resting activity levels in the auditory system. Hypothesis: Conductive hearing loss will change spontaneous activity levels at the inner hair cell synapse, and lead to auditory deprivation of central auditory pathways. Experiments: In a mouse model, a 50-60dB conductive loss was produced by blocking the ear canals. Resting neural activity patterns were quantified in cochlear nucleus and inferior colliculus using c-fos immuno-labelling. Conductive hearing loss subjects were compared to normal hearing controls and subjects with bilateral cochlear ablation. Results: Subjects with conductive hearing loss showed a trend in reduction in c-fos labelled cells in cochlear nucleus and a more significant reduction of labelled cells in the central nucleus of inferior colliculus compared to normal controls. Conclusions: There is a reduction in resting neural activity in auditory brainstem and midbrain as a result of conductive hearing loss. Such changes may influence the developing auditory brain during early postnatal years when the system is highly plastic.

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Acknowledgements

I would first like to thank my supervisor Dr. Robert Harrison for his guidance, support and the opportunity to study and benefit from his mentorship during my time with him. It has been a pleasure working in his lab and gaining knowledge in his field of expertise. I would also like to thank the members of my supervisory committee, Dr. Karen Gordon and Dr. Paul Frankland, who offered many helpful suggestions and gave me the benefit of their knowledge throughout this Masters. I am grateful to the technicians at the University of Toronto microscopy laboratory, Steven Doyle and Battista Calvieri for teaching and guiding me in using the Scanning Electron Microscope. I would also like to thank Michael Woodside and Paul Paroutis in the imaging facility at the Hospital for Sick Children for their help in using the epiflorescent microscope and the software of analysing my results. I would also like to thank the members of our lab, Dr. Trecia Brown, and Lisa D‟Alessandro for their positive reinforcement, support and enthusiasm for research which has made working each day in the lab an exciting learning experience. This research was funded by a Canadian Institutes of Health Research (CIHR; grant VIH105441) and by the Masonic Foundation of Ontario. Lastly, I would like to thank my family and friends, especially my husband, Binit Negandhi for his enduring support and always rejoicing in my success.

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Table of contents Abstract ................................................................................................................................ ii Acknowledgements .............................................................................................................. iii Table of contents .................................................................................................................. iv List of Abbreviations ............................................................................................................. ix 1 CHAPTER: INTRODUCTION .................................................................................................. 1

2

1.1

Research Objectives ............................................................................................................1

1.2

Thesis Outline .....................................................................................................................2

CHAPTER: BACKGROUND ................................................................................................ 3 2.1 2.1.1 2.1.2 2.1.3

2.2 2.2.1

3

Anatomy and Physiology of the Mammalian ear ..................................................................3 The Inner Hair Cell ...................................................................................................................................7 The Outer Hair Cell ..................................................................................................................................9 Ascending Auditory Pathways ...............................................................................................................10

Conductive Hearing Loss .................................................................................................... 17 Otitis Media ...........................................................................................................................................19

2.3

Spontaneous/Resting Activity ............................................................................................ 22

2.4

Plasticity in the auditory system ........................................................................................ 24

2.5

Hypothesis ........................................................................................................................ 26

CHAPTER: EXPERIMENTAL METHODS ............................................................................ 27 3.1

The Mouse Model ............................................................................................................. 27

3.2

Experimental Design and Procedure .................................................................................. 27

3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.2.6

ABR Measurements ...............................................................................................................................31 Tissue Preparation .................................................................................................................................32 C-fos Labelling ........................................................................................................................................33 NeuN Labelling .......................................................................................................................................35 Method of Quantification ......................................................................................................................36 Scanning Electron Microscopy ...............................................................................................................38

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CHAPTER: EXPERIMENTAL RESULTS ............................................................................... 39 4.1

ABR Results ....................................................................................................................... 39

4.2

SEM Results ...................................................................................................................... 40

4.3

RESULTS: Immunocytochemistry........................................................................................ 41

4.3.1 4.3.2 4.3.3

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Cochlear Nucleus ...................................................................................................................................41 Inferior Colliculus ...................................................................................................................................42 Percentage of c-fos active neurons in the brainstem and midbrain ......................................................45

CHAPTER: DISCUSSION .................................................................................................. 48 5.1

Discussion of experimental protocol & results.................................................................... 48

5.2

Clinical significance of results ............................................................................................ 54

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CHAPTER: CONCLUSIONS & FURTHER DIRECTIONS ......................................................... 56

APPENDIX 1 ......................................................................................................................... 66 1.

c-FOS/NeuN Immuno-labelling Protocol ............................................................................... 66

2.

Scanning Electron Microscopy sample preparation ................................................................ 67

3.

Results: Immunocytochemistry in the IC and CN .................................................................... 71

4.

ABR Results: after removal of conductive block ..................................................................... 73

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List of Figures Figure 2-1 the human ear showing the outer, middle and inner ear. Adapted from Neuroscience Second Edition, D. Purves et al. 2001, Pg 280. ......................................................................................................... 4 Figure 2-2 Cross-section through the cochlea. Image adapted from Neuroscience Second Edition, D. Purves et al. 2001. Pg 284. ............................................................................................................................................ 4 Figure 2-3 Movement of basilar membrane creates a shearing force that bends the stereocilia of the hair cells. Image adapted from Nature Reviews in Neuroscience. ...................................................................... 6 Figure 2-4 Mechanoelectrical transduction mediated by hair cells. Image adapted from Neuroscience-second edition, D. Purves et al. 2001, Pg 280 ........................................................................................................... 7 Figure 2-5 The role of the ribbon synapse in neurotransmission from the inner hair cells to afferent nerve fibres. ............................................................................................................................................................ 8 Figure 2-6 Schematic representation of the ascending auditory pathway in the mammal. Input from one ear is depicted. Adapted from The Biology of Hearing and Deafness, Harrison RV, 1988. DCN - dorsal cochlear nucleus; AVCN - anteroventral cochlear nucleus; PVCN posteroventral cochlear nucleus; MTB - medial nucleus of the trapezoid body; MSO – medial nuclues of the superior olive; LSO – lateral nucleus of the superior olive; C of P – commissure of Probst; C of IC – commissure of inferior colliculus; PN – pericentral nucleus; CN – central nucleus; EN – external nuclus; VN – ventral nucleus; MN – medial nucleus; DN – dorsal nucleus; CC – corpus callosum ................................................................... 13 Figure 2-7 The human auditory cortex. The primary auditory cortex is shown in blue and the surrounding areas of auditory cortex are shown in pink. Image adapted from Neuroscience – second edition, edited by D. Purves et al. 2001; Pg 294. ................................................................................................................ 16 Figure 2-8

Image showing a middle ear infection (Otitis media). Image adapted from website:

functionalmedicinesolutions.com ............................................................................................................... 20 Figure 2-9 Difference between a normal tympanic membrane to one affected by an ear infection (Otitis Media). Image adapted from Otitis Media – A pocket guide by Hawke M. 1994. .................................... 21

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Figure 3-1 Schematic of the ear showing the area in the external ear canal that was blocked using dental cement. ....................................................................................................................................................... 29 Figure 3-2 Auditory Brainstem responses to 32 kHz tonal stimuli. Each ABR waveform reflects the average response from 512 sweeps presentations. Responses are presented in descending order from 90 to 10 dB SPL. Waveforms are clear and reproducible down to 20dB SPL (*). ....................................................... 32 Figure 3-3 Image of c-fos fluorescently labelled active neurons as seen under a microscope. Images viewed at 1X and 20X resolutions. ............................................................................................................................. 34 Figure 3-4 NeuN stained tissue as seen under the microscope. Images viewed at 1X and 20X resolutions. ..... 36 Figure 3-5 Screen print showing method of analysis when quantifying labelled neurons. ............................... 37 Figure 4-1 ABR threshold elevations (50-60dB) in mouse model of conductive hearing loss (CHL). Deaf subjects had no measureable ABR’s even at intensities over 100dB. ........................................................ 39 Figure 4-2 Inner and Outer hair cells show normal morphology after induced conductive loss. ..................... 40 Figure 4-3 Results at the cochlear nucleus indicate a trend of reduction in resting activity levels after induced conductive loss. ANOVA *(p=0.007). Pair-wise (t-test) comparisons of Control vs CHL (p=0.195), CHL vs Deaf (p=0.004) and Control vs Deaf (p=0.051). ..................................................................................... 42 Figure 4-4 Results at central nucleus of IC show a significant reduction in c-fos labelled neurons **(F(2,15)=13.005, P=