This thesis addresses the pressing concern of clinical drug-induced hearing loss (ototoxicity). Described herein is the mechanism by which ototoxicity emerges following drug administration both in a clinical setting and in an in vitro model assay system used for its investigation, through use of mouse cochlear cultures. The predominant nature of this research concerns the identification of novel otoprotectants - compounds that when co-administered alongside clinical drug treatments can prevent the unfortunate ototoxic side-effect from occurring. Here I present my research, focussing on the identification of a number of novel compounds that have the potential to be taken forward to in vivo screening and, ultimately, clinical trials. Moreover, for each identified compound I present my investigation of their mechanism of protection - which could arise either by preventing the entry of the ototoxicity-inducing drugs into the sensory hair cells of the inner ear, or prevent their induction of apoptosis once inside the cell. To investigate their protective mechanism I employed a variety of methods, including: electrophysiology, fluorescent imaging and mitochondrial respirometry. Conclusively, I have identified at least five novel otoprotectants with the potential for clinical use. I show that three of these compounds likely block the entry of the damaging drugs into sensory hair cells, whereas the remaining two are thought to work intracellularly. Moreover, the two most effective compounds that I have identified seemingly work intracellularly, suggesting this to be the most viable mechanism of otoprotection for further investigation. I also show the potential for compound modification based on their mechanism of protection as a way of improving a compound's otoprotective profile. Lastly, I devised an assay for the screening of clinical drug effects on mitochondria and employ this as a new avenue of screening for otoprotection.

Transforming sound vibrations into neural signals, the process of mechano-electrical transduction (MET), is achieved via the movement of rows of stereocilia located atop cochlear hair cells, activating non-selective MET channels. Recording MET currents using whole-cell patch clamp electrophysiology has allowed for various investigations centred on the MET channel. There is a statistically significant difference in MET current amplitudes between mid-apical and mid-basal outer hair cells. The larger MET current, flowing through basal hair cells explains a key contributory element to their increased susceptibility to the ototoxic effects of aminoglycoside antibiotics, as they are thus able to enter these cells more readily, in greater abundance. The cell penetrating peptides D-TAT and D-JNKi1 have been studied in cochlear hair cells. They are both high affinity, voltage dependent, permeant blockers of the MET channel in inner (IHCs) and outer hair cells (OHCs). Whilst ototoxic at higher concentrations, they may have therapeutic potential at lower concentrations, providing ototoprotection against aminoglycoside induced ototoxicity by competitively blocking the MET channel. There is an apical to basal gradient in the OHC MET channel block caused by D-TAT and D-JNKi1, the affinity of the block increasing from apex to base. Compellingly, the first evidence of a gradient in the MET channel properties of IHCs is presented here, a gradient in D-JNKi1 block. Properties of an anomalous mechano-sensitive current, elicited by inverse stimuli relative to the MET current, have been elucidated via the use of the compounds dihydrostreptomycin (DHS) and FM1-43. When compared with the MET current, DHS block of the anomalous current is two orders of magnitude less strong and FM1-43 block has altered voltage dependence. TRPC3/6 knock-out mice, whilst previously thought to have a basal OHC specific MET deficit, have been shown to exhibit normal MET currents. In addition, so too have TRPC1/3/6 and TRPC1/3/5/6 knock-out mice.

The stapedius muscle, which is located in the middle ear, goes into contraction when the ear is exposed to high sound intensities. This muscle activation is called ‘the acoustic reflex’. Measurement of the acoustic reflex is clinically of importance since it can reveal diagnostic information about the middle ear’s pathologies. Moreover, this muscle-activation alters the acoustic characteristics of the middle ear (i.e. the acoustic impedance and the power reflectance), which in turn, can significantly manipulate one’s perception of sounds. In the present study, these acoustic characteristics are measured in the ear canal by means of absorbance measures using equivalent Thevenin circuit theory. The quantities are then compared to form the shift responses between the baseline (before the activation) and the post-activator effect. This project investigates the shifts in power reflectance and admittance of the middle ear caused by the stapedius-muscle contraction. The wideband characterization (0.1- 8 kHz) of these acoustic reflex-induced shifts is achieved using chirp signals as a probe and through ipsilateral broadband noise activator. The data acquisition and signal processing of the project are carried out using MATLAB software. The hardware consists of National Instruments USB-6212 data acquisition interface and low noise microphone system Etymotic Research ER-10B+. A group of 10 adults including 5 males and 5 females are recruited as the participants for the project. The measurements of the reflectance shifts indicate that the most robust frequency region affected by the acoustic reflex is up to 4 kHz whereas for the admittance shifts, this region is up to 2 kHz. In addition, it is shown that the stapedius-muscle contraction leads to the attenuation of the lowfrequency transmission into the middle ear (less than 1 kHz) consistent with a stiffnesscontrolled system in this range of frequencies. In contrast, the results imply that the activation of the stapedius muscle leads to a slight enhancement of the frequency transmission in the range of 1-4 kHz (corresponding to the speech frequency band). These findings suggest a beneficial role for the stapedius-muscle contraction in the perception of speech during vocalization. Furthermore, the implemented methods in this project  can be useful in better understanding the effect of the stapedius-muscle contraction on the speech perception both in normal hearing and hearing impaired persons.