In the developing mammalian cochlea, the inner hair cells (IHCs) transiently and the outer hair cells (OHCs) permanently receive efferent innervation originating in the auditory brain stem. This cholinergic input is mediated by a9/a10nicotinic acetylcholine receptors (nAChRs) and is inhibitory due to the subsequent activation of calcium-dependent potassium channels. In the mature cochlea, efferent inputs modulate OHCs activity, thereby significantly influencing sensitivity and frequency selectivity of the cochlea, and most likely playing a role in sound protection. During development, IHC efferents are thought to affect auditory nerve fiber activity and thereby possibly influencing maturation processes in the auditory pathway. For efferent inputs to function adequately, efferent synapses need to be set up properly during development. Here we propose that in addition to a9/a10 nAChRs, the a1 nAChR, a subunit found in muscle, is functional in hair cells and may play a role in efferent synapse formation and/or maintenance. This hypothesis is based on our finding that in addition to the response mediated by a9/a10nAChRs, in developing mouse IHCs a nicotine-mediated response exists that is inhibited by a blocker specific for the a1 nAChR.
In Aim 1 the newly found nicotine- mediated response will be characterized. Since a9/a10nAChRs do not respond to nicotine, this implies the existence of other nAChRs. In acutely excised mouse cochlear preparations, patch-clamp recordings will be performed from IHCs to determine the timeline of expression of the nicotinic-sensitive current. We will use nAChR subtype selective pharmacological agents to identify subunits mediating the response and to further test the hypothesis that the a1 nAChR subunit is part of the receptor. To test if the nicotine-sensitive nAChR is involved in efferent synaptic transmission, we will measure whether efferent synaptic currents are partially mediated through this receptor and we will study whether its subcellular localization coincides with efferent postsynaptic structures using specific fluorescent toxins. Additionally, the presence of a nicotinic-sensitive response will be tested in OHCs.
In Aim 2, we will use a mouse model lacking a1 nAChR expression in the inner ear, to test for its possible function. We will test whether the nicotine-mediated response is lost in the absence of a1. If so, we will conclude that a1 is a key element of the nicotine-sensitive AChR. Immunohistochemistry and labeling with fluorescent toxin markers will be used to test if the lack of the a1 nAChR is associated with changes in efferent innervation pattern. Using electrophysiological measures, we will investigate whether functional efferent synapses develop normally in the absence of a1. A potential role in synapse maintenance will also be examined. If a role for the muscle type a1 nAChR is confirmed for hair cell efferent synapse formation/maintenance, this will provide an important key for further deciphering the under- lying molecular mechanisms. The extensive characterization of synapse formation and maintenance at the neuromuscular junction can then aid our understanding on how these processes might work in the inner ear.

Public Health Relevance

Hair cells in the mammalian cochlea receive efferent innervation from the auditory brain stem that modulates hair cell activity and thereby improves our hearing capabilities. For efferent inputs to function adequately, efferent synapses need to be set up properly during development. Here we propose to study mechanisms involved in efferent synapse formation, specifically the composition and localization of acetylcholine receptor subunits during synapse formation that we propose may be crucial in this process.

National Institute of Health (NIH)
Small Research Grants (R03)
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Special Emphasis Panel (ZDC1)
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Cyr, Janet
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Johns Hopkins University
Schools of Medicine
United States
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