Vestibular hair cells are mechanotransducing sensory receptors in the vertebrate inner ear that detect movement and orientation of the head with respect to gravity. The morphologies of their ciliary bundles vary greatly for different species, endorgans, and within the same endorgan. Bundle morphology in the turtle utricle, like other species, demonstrates highly organized regional variations. These structural differences in bundles impact their mechanical behavior and the process of mechanotransduction. To further understanding of the mechanical behavior of hair bundles, this work experimentally measured the stiffness of bundles with differing morphology, the stiffness contribution of interciliary links and the mechanical properties of the kinocilium in the turtle utricle. The stiffness of hair bundles of varying structure and location along a medial to lateral transect of the utricle was examined. Bundle stiffness was greatest in the striola and demonstrated a systematic decline with location from the line of polarity reversal. The average stiffness of bundles in the striola and extrastriola were 82 ± 46 (n=48) and 9 ± 5 (n=25) µN/m, respectively. The stiff and weak bundles demonstrated characteristic morphologies. The stiffest bundles have short kinocilium, tall stereocilia, and ratios of kinocilium to tallest stereocilia height (KS) close to 1. In contrast, the compliant bundles have tall kinocilium, short stereocilia, and KS ratios ranging from 1.6 – 8. The stiffer bundles also tend to have longer array lengths and steeper slopes. Measurements of bundle stiffness in the turtle utricle are lower than those previously reported which may be attributed to morphological differences between species. The stiffness contributions of the interciliary links were also examined through their selective removal with exposure to the Ca²⁺ chelator BAPTA and the protease subtilisin. BAPTA treatment reportedly breaks tip, kinocilial and ankle links while subtilisin breaks the shaft and ankle links. Following BAPTA and subtilisin treatments, bundle stiffness reduced by 65 ± 10% and 63 ± 11%, respectively. The mechanical properties of the kinocilium were measured with novel techniques. Flexural rigidity (EI) was measure while the kinocilium was fixed at the height of the tallest stereocilia using a glass supporting probe. Through both force deflection and a high speed video technique, measured values of EI ranged from 1460 – 6150 pN·µm2. The rotational stiffness of the kinocilium about its apical insertion was also measured. Bundles were treated with BAPTA to break the kinocilial links and separate the kinocilium from neighboring stereocilia. Using a force deflection technique, the rotational stiffness of the kinocilium was measured as 120 ± 17 pN·µm/rad. / Ph. D.

The ear is the organ used for hearing and maintaining equilibrium. In the inner ear, the vestibular system is responsible for the sense of balance. The main organs of the vestibular system are the semicircular canals, the saccule, and the utricle. Within each of the vestibular organs, sensory receptors in the form of hair cells detect motion and send a message to the brain for interpretation. Hair cells found in different parts of the inner ear are structurally different and are mechanically specialized to perform different functions. In this study, the linear and torsional stiffnesses were measured for hair cells located in the red ear slider turtle utricle. The system used to measure the stiffnesses was composed of a glass whisker (attached to a pipette) used to produce a force on the tip of the bundle, an extrinsic Fabry-Perot interferometer (EFPI) to measure the displacement of the pipette, and a photoelectronic motion transducer (PMT) to measure the displacement of the bundle. Using the measured values of whisker stiffness, whisker displacement, and bundle displacement, the stiffness of the bundle was calculated using statics. For each bundle tested, the location of the bundle was determined by measuring its position from a landmark in the utricle, the line of polarity reversal, characterized by a 180o change in direction of the hair bundles. Stiffness results showed that the linear stiffness of a bundle increased in the area surrounding the line of polarity reversal, otherwise referred to as the striolar region (average linear stiffness of 2.27 E-04 N/m). The average linear stiffness value of bundles found lateral to the striolar region was 6.30 E-05 N/m and in the region medial to the striolar region was 1.16 E-04 N/m. A wide range of linear stiffnesses were found in hair cells medial to the striolar region. There was no correlation found between the torsional stiffness of a bundle and its position and the height of a bundle and its linear or torsional stiffness. As the force applied to a hair bundle was increased, the measured linear stiffness of the bundle also increased. / Master of Science

The vestibular system consists of several organs that contribute to ones sense of balance. One set of organs, otoconial organs, have been shown to respond to linear acceleration (1949). Hair bundles (and hair cells), which are the mechano-electric transducers found within otoconial organs, respond to displacement of the overlying otoconial membrane (OM). Structure, position and orientation of the OM within the head may influence the stimulus of hair bundles by changing the deformation characteristics of the OM. Therefore, studying the deformation characteristics of the OM with finite element models presents a unique advantage: the ability to study how different variables may influence the deformation of the OM. Previous OM models have ignored complicated OM geometry in favor of single degree of freedom (De Vries 1951)or distributed parameter models (Grant et al. 1984; Grant and Cotton 1990; Grant et al. 1994). Additionally, OMs have been modeled considering three dimensional geometry (Benser et al. 1993; Kondrachuk 2000; 2001a), however OM layer thicknesses were assumed to be constant. Further, little research has investigated the effect of position and orientation of otoconial organs on the deformation of the OM (Curthoys et al. 1999), due to natural movement of the head. The effect of structure, position and orientation of the utricle of a red ear slider turtle on the stimulation of hair bundles in the OM is investigated here. Using confocal images, a finite element model of the utricle OM is constructed considering its full 3D geometry and varying OM layer thickness. How specific geometric variables, which are missing from other OM models, effect the deformation of the utricle OM is studied. Next, since hair bundles are part of the structure of the OM, their contribution to the deformation of the utricular OM is quantified. Then, using computed tomography of a turtle head and high speed video of turtle feeding strikes, acceleration at the utricle during natural motion is estimated. Finally, the effects of orientation of the utricle in the head on the stimulus of hair bundles within the organ is investigated. In summary, a model and methods are developed through which deformation of the turtle utricle OM through natural movements of the head may be studied. Variables that may contribute to utricle OM deformation are investigated. Utricle OM geometry, hair bundles, position and orientation all play a role in utricle OM deflection and therefore hair bundle stimulus. Their effects are quantified and their roles are discussed in this dissertation. / Ph. D.

The vestibular system is one of our main organs responsible for the sense of balance. This system is located within the inner ear and contains cells with ciliary bundles. These hair cells are transducers that convert a mechanical movement, detected by the bundle of cilia extending from their top surface, into an electrochemical signal to be sent to the brain. The bundles vary structurally within the organs of the inner ear, and this structural difference may play a role in the mechanical properties of each bundle. Analyzing the mechanical properties of the cells will provide information necessary for understanding the transduction process. In an effort to evaluate one of these properties, cell bundle stiffness, a system was designed to mechanically stimulate the bundles within their physiological range and then measure the resulting displacement. The mechanical stimulation was the result of a force applied to the tip of a bundle with the end of a glass whisker. The distance the base of the whisker moves is measured by an extrinsic Fabry-Perot interferometer (EFPI). The magnitude of this movement is compared with the amount the bundle is deflected, detected by a photoelectronic motion transducer (PMT). Knowing these displacements and the stiffness of the glass whisker, simple kinematics is used to determine the bundle stiffness. System tests were conducted on imitation bundles (whiskers of known stiffness) and the experimental stiffness differed from the known value by less than 4.5% for every test. These results lead us to conclude the system was in good working order and could be used to conduct tests on cell bundles. For tissue tests, this work focused on the hair cells located within the utricle, which senses linear accelerations of the head. Within the utricle, we examined two types of hair cells: non-striolar (medial type II) and striolar. Tests on twelve medial type II cells found bundles ranging in stiffness from 0.26 to 2.62 x 10⁻⁵ N/m. Results with striolar bundles provided a range from 2.83 to 27.10 x 10⁻⁵ N/m. The results of the preliminary tissue tests lead us to conclude that the average stiffness of the striolar and non-striolar bundles seems to vary by an order of magnitude. This is consistent with the relative relationship produced through a computer model. However, the model predicted larger stiffness values for both types of cells. / Master of Science

Background Hair cells (HCs) are the sensory receptors in both the auditory and the vestibular organs of the inner ear. Supporting cells (SCs) are non-sensory cells embracing the HCs. Injuries of the HCs by aging, acoustic trauma or ototoxic drugs (mainly aminoglycosides, e.g. gentamicin) and cisplatin, often cause permanent impairment of hearing and balance. Birds and amphibians can regenerate their auditory and vestibular HCs after injury through proliferation of SCs or direct transdifferentiation of a SC into a HC. For mammals this ability is limited and spontaneous HC regeneration occurs only in the vestibular sensory epithelia. The utricle is one of the five vestibular organs and contributes to our balance by registering linear acceleration and head tilts. The aim of this PhD thesis was to investigate morphological and morphometric events during spontaneous HC regeneration following gentamicin exposure in neonatal rat utricular explants. Methods Long-term organ culture of macula utriculi, which is stable and reproducible for up to 28 days in vitro (DIV), was used in all papers in the thesis. HC damage was induced by gentamicin. On 2 DIV the explanted utricular maculae were divided into two groups, a control group and a gentamicin-exposed group. In the latter group macular explants were exposed to gentamicin for 48 hours during 2-3 DIV and then allowed to recover. Morphologic and morphometric evaluations were done from utricles harvested at various time points during 28 DIV. Imaging techniques used were light microscopy, including immunohistochemistry, and transmission electron microscopy. Results In the control group the epithelia were well preserved with a slight decline in HC density after 14 DIV. In the gentamicin-exposed group there was an initial substantial decline in HC density and thereafter the proportion of HCs in relation to SCs increased significantly. Using BrdU as a proliferation marker and myosin 7a as a HC marker, we found no cells that were double marked. At the ultrastructural level, the apical occlusion of the explanted epithelia was intact in both the control and the gentamicin exposed group during the entire in vitro period. Cells that seemed to be in a transitional state, transforming from SCs into HCs were observed in the gentamicin-exposed group. These cells had cytoplasmic extensions basally i.e. foot processes, an assembly of mitochondria basally in the cell or in these foot processes, and often apical SC extensions covering the HC. HCs classified as transitional cells had an increased number of SC connections to their basal parts compared to mature HCs. Conclusions  In these neonatal rat utricular explants: - The morphological structure of the sensory epithelia was well preserved during long-term culture. - The renewal of hair cells after gentamicin exposure occurred through direct transdifferentiation of supporting cells into hair cells. - There was also a proliferative response by the supporting cells, but this supporting cell proliferation did not contribute to the generation of new hair cells. - Cells in a transitional state, showing a characteristic morphology, were observed during the process of transdifferentiation from supporting cells into hair cells. - The tight junctional seal of the epithelia stayed morphologically intact also after gentamicin exposure. - Gap junctions were observed in between supporting cells but not found in between hair cells and supporting cells or between transitional cells and supporting cells.

Clinical ototoxicity - permanent hearing loss caused by medications - is estimated to affect millions of patients annually. Two classes of drug are largely to blame: platinum-based chemotherapeutics, primarily cisplatin, and aminoglycoside antibiotics. Development of methods to prevent ototoxicity depends upon an understanding of its mechanisms and may benefit from an understanding of native protective pathways of the inner ear. As the mechanisms behind cisplatin ototoxicity remain unclear, I first sought, and herein report, a refined mouse model of cisplatin ototoxicity which will allow for further in vivo investigation of cisplatin ototoxicity and potential methods for its prevention. This low-dose, multi-cycle model was found to accurately reproduce cisplatin ototoxicity as it has been described clinically and histopathologically. I then used this mouse model of cisplatin ototoxicity to investigate cisplatin pharmacokinetics in the cochlea and their role in driving cisplatin ototoxicity. Cisplatin was found to be retained within the cochlea for months following its administration. This initial finding in mice was extended to cochlear tissue samples from deceased human patients. Analysis of intra-cochlear cisplatin distribution in murine and human tissue identified the stria vascularis region of the cochlea as a promising target for intervention. With the nature of aminoglycoside ototoxicity better understood, I investigated a native inner ear protective pathway which could be leveraged to promote sensory hair cell survival. The improved hair cell survival that has previously been demonstrated as a result of heat stress was found to be mediated by cell-cell communication via extracellular vesicles. Further, hair cell protection against aminoglycosides could be reproduced through the application of exogenous, non-inner ear-derived extracellular vesicles. In sum, these data provide new insight into mechanisms of ototoxicity and details of cellular pathways which can help protect against it.

This study reports the occurrence of daily rings in the otoliths of Oncorhynchus nerka fry and examines their relationship to growth. In experiment 1, sockeye salmon fry were collected from the Fulton River spawning channel at Babine Lake, British Columbia in May 1978. The fish were reared for 26 days in enclosures in the spawning channel and were sampled every seven to ten days. Sagittae were removed from 25 fish from each sample, and the growth rings in one otolith from each fish were counted. A regression of the number of rings on the number of days since capture showed that these rings are, on average, formed daily, beginning at the time of emergence. A number of possible technical and biological causes of variation in ring counts within and between samples are considered. In Experiment 2, sockeye salmon fry were reared in the laboratory from fertilized eggs taken in the fall of 1978 at the Weaver Creek spawning channel near Mission, British Columbia. A random sample of 64 of these fry was marked to enable identification of individuals. Each individual was weighed initially on June 6 or 8, again on July 6, and surviving fish were weighed a third time on July 20. After a final weighing, sagittae were removed and a standard otolith radius was determined by counting back the appropriate number of daily rings which corresponded to each weight. The regression of £n otolith radius on £n fish weight was linear, and had an R2 of 0.92, which demonstrates a relationship between the mean width of a daily ring in sockeye salmon fry sagittae, and a mean daily change in the weight of the fry. Using this regression line, we back-calculated the previous weight of the individual fish from the corresponding otolith radius and a latter fish weight and otolith radius and found the errors to be relatively small — in the order of 15 per cent. / Science, Faculty of / Zoology, Department of / Graduate

Within the vestibular system are otolith organs, both the utricle and saccule. The primary function of these organs is to transduce linear head accelerations and static head tilts into afferent signals that are sent to the central nervous system for the utilization of image fixation, muscle posture control, and the coordination of musculoskeletal movement in dynamic body motion. The utricle of the red ear slider turtle was studied in this dissertation. The turtle's utricle is composed of several layers. The base layer contains a set of neural receptor cells, called hair cells, and supporting cells. The three layers above the base layer compose the utricle's otoconial membrane (OM) and are: 1.) a saccharide gelatinous layer, 2.) a column filament layer, and 3.) a calcite and aragonite otoconial crystal layer. The primary goal of this research was to study the dynamic response of the turtle's OM to a variety of natural inertial stimuli in order to characterize its inherent mechanical properties of natural frequency ("n), damping ("), and shear modulus (G). The medial-lateral (ML) and anterior-posterior (AP) anatomical axes parameters were measured for the utricle. The ML axis median with 95% confidence intervals was found to be "n = 374 (353, 396) Hz, " = 0.50 (0.47, 0.53), and G = 9.42 (8.36, 10.49) Pa. The AP axis median with 95% confidence intervals was found to be "n = 409 (390, 430) Hz, " = 0.53 (0.48, 0.57), and G = 11.31 (10.21, 12.41). Nonlinearites were not found to occur in the OM for the tested inertial stimuli and no significant difference was found between the mechanical properties for the ML and AP axes. Additionally, this research presents the initial steps to form a novel bio-inspired accelerometer based on the morphology of the utricle. The primary transducer element for this possible otolith organ inspired accelerometer design is a droplet interface bilayer (DIB). A DIB is a lipid bilayer that is formed when the interface of two aqueous droplets, that contain free-floating lipids, are joined. The aqueous droplets are suspended in a nonpolar environment (oil) and the oil/water interface forms a lipid monolayer. This research developed and used an experimental test setup to characterize the mechanoelectrical characteristics of a micron-sized DIB. This information, along with examples in the text, could be used to further design the aforementioned accelerometer. / Ph. D.

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