3D ultrastructural analysis of the subcellular organisation of inner hair cells and of their innervation during ageing.

Hearing deteriorates progressively with age. This results from dysfunction of the cells in the cochlea of the inner ear that detect sound (the sensory cells, so-called "hair" cells), and/or the nerves that carry the signals to the brain. The hearing deficits are permanent. An early manifestation is difficulty in understanding speech in noise, then reduced ability to hear quieter and higher frequency sounds that progressively worsens with decreasing sensitivity to louder sounds and lower frequencies. An estimated 70% of those over retirement age suffer some form of hearing impairment. It is a major factor in reducing their quality of life. Amelioration of the condition is currently limited to technological solutions such as hearing aids and cochlear implants. There are no drug treatments. In order to improve the effectiveness of the technologies and to discover pharmacological targets for treatments, a better understanding of the progressive changes to the hair cells and nerves that occur with ageing is essential.
Recent work has suggested that a major factor contributing to the deterioration of hearing with age is a slowly progressive loss, beginning quite early in life, of the nerve fibres that normally transmit sound information from the cochlea to the brain (afferent fibres), and which connect with the sensory cell - the inner hair cell (IHC) - that converts sound signals into the electrical signals that stimulate the nerves. There may also be re-organisation of the nerves ("efferent" fibres) that carry information from the brain to the afferent nerve terminals to modulate the auditory signals reaching the brain. In addition there is evidence of changes to the internal organisation of the IHCs themselves. In our current research we have applied two techniques for three-dimensional electron microscopy (3DEM) to characterise of the internal structures of IHC and the distribution of the contacting nerves fibres around it. With these techniques, we have demonstrated a previously unrecognised degree of structural organisation in IHC and have been able to map the distribution of all the afferent nerve terminals around the cell and the internal organisation, a feat not previously accomplished but made possible by the application the 3DEM technologies. We have found a continuous network of cellular membranes and organelles that appears to be structurally related to the synaptic machinery of the cell (the interface between the cell and the nerve where signals are transmitted) and to the position of the nerve terminals. Preliminary work has also shown disruptions to intracellular organisation of IHC that appear to be commensurate with loss nerve fibre loss during ageing. In the proposed project we will extend these techniques to fully characterise age-related changes to the cells and their innervation. We will also develop the use of a third 3DEM technique, array tomography, which offers the potential for routine 3D analysis of tissues without the need for the specialised microscopes that the other two techniques require. The central aim of the project is to map at the cellular and sub-cellular levels the changes to the internal structures of IHCs in the ageing cochlea and to examine the relationship between those and the loss of afferent terminals. We will also identify the compensatory changes to IHC and to the remaining afferent nerves, and the re-organisation of the efferent nerves. The project will provide a comprehensive assessment of the cellular basis of the deterioration of hearing with age.
This application also will bring together researchers with expertise in electron microscopy and 3D analysis, cochlear anatomy, cochlear physiology and the molecular and genetic bases of deafness. This co-ordinated approach is designed to generate new approaches and future collaborations to further research into the life-course of the auditory system.

Hearing deteriorates progressively with age. Recent work has suggested that a major contributory factor is a slowly progressive, systematic loss, beginning early in life, of the afferent terminals that normally surround each inner hair cell (IHC) in the mammalian cochlea. The main aim of this project is to undertake a comprehensive 3D, ultrastructural analysis of the age-related changes to the intracellular organisation and synaptic machinery of IHC and their relationship to the progressive loss and re-organisation of the afferent and efferent terminals that occurs over the life of the animal.

In our current BBSRC-supported project, we have applied two techniques to explore the 3D structural organisation of IHCs and their innervation. We have been able to define the distribution of afferent terminals in unprecedented detail and have revealed a previously unrecognised organisation of the infranuclear region of the IHC, comprising of a network of intracellular membranes, mitochondria and associated vesicles that appears to be related to the distribution of ribbon synapses and afferent terminals. Preliminary work from our laboratory has suggested that this network may deteriorate with age. We now propose to use these techniques, serial block face scanning electron microscopy (SBF-SEM) and electron tomography to detail the progression of structural changes and re-organisations at the IHC and its innervation that occur with ageing. We shall use two different strains of mouse, one which retains good hearing for at least 2 years, and one that is used as a model of early onset hearing loss. We also propose to develop and apply a further technique for 3D electron microscopy, array tomography, which has the advantage of enabling multiple imaging of the same section series at different magnifications.

The project will provide a detailed understanding of the cellular basis of age-related deterioration of hearing.

Who will benefit?
1. People with age-related hearing loss
2. Clinical professionals
3. UK and other companies producing cochlear prostheses
4. The wider public.

How will they benefit?
Age-related hearing loss (ARHL) is a major disabling condition, affecting the quality of life of elderly people. More than 70% of people over retirement age have hearing loss sufficient to impair normal communication. By contributing to a detailed understanding of the subtle pathologies of hair cells in the earliest stage of progressive ARHL this project will lay foundations for addressing the fundamental bases of auditory deficits. Thus, in the longer term the project will contribute to determining potential pharmaceutical targets of intervention or other means to alleviate these conditions, thereby improving the quality of life for elderly people, and relieving the economic and social burden that they impose, with benefits to the wider public.
In the shorter term, the results of the project will be beneficial clinically in enabling doctors and health workers to inform their patients better about the nature of their disease. The close association of the Ear Institute with the Royal National Throat Nose and Ear hospital provides a conduit for bringing the results of the scientific research to the attention of the clinical community. Professors Forge and Ashmore have professional relationships with this group and are regular speakers to interested professional and patient groups about their research. Companies producing cochlear implants, devices inserted into a deaf ear that can partially restore hearing, are also interested in the fundamental bases of cochlear pathologies that disturb auditory function. This knowledge enables improvements in auditory signal processing strategies to overcome the deficits and widen the candidature for implantation including to those with ARHL. We are already working with one such company to explore preservation of residual hearing after implantation and have contacts with others. These relationships afford opportunities for direct knowledge transfer.
The project will also have impact more widely in cell and structural biology. 3D ultrastructural analysis techniques have not been widely applied and the methods and procedures developed in this project will provide protocols for more widespread application of the emerging technologies, in particular for array tomography which has the potential for more routine application.
The project will also provide an opportunity to retain a talented young scientist with unique skills, Dr Bullen the researcher co-investigator on this application, in the field of auditory research where there is a shortage of young investigators. It will enable her to begin to establish her independent research career, and to cement mutually beneficial collaborations with other laboratories working in related fields. In addition, the project affords opportunities for training in the use of software for manipulation of large data sets which will be exploited through work experience placement of school students and summer studentships for undergraduates and others in established programmes at the Ear Institute and the department at Birkbeck College.
It is important that the results of the work are communicated to the general public. As President of the Physiological Society Prof Ashmore has a remit in communication of science to the public. Dr Moores has a proven track-record of public communication of science. She was the 2006 winner of the prestigious DeMontfort medal for science communication (SET for Britain). Prof Forge and Prof Ashmore have given interviews about hearing and deafness to BBC radio and are regularly involved in presenting their work at events organised by hearing research charities, to whom they also act as advisers. Similar means of communication, with Dr Bullen involved, will be developed during the course of project.