This is a competing renewal application to continue study of the mechanism of cholinergic inhibition of cochlear hair cells using biophysical and molecular genetic analyses. We will also examine the developmental regulation of this response. Efferent axons arising from cells in the brainstem provide an inhibitory input to outer hair cells in the mammalian and avian cochleas. This inhibitory effect is thought to result from the release of acetylcholine (ACh) from the efferent endings. Several lines of evidence point to the fact that the cholinergic response of cochlear hair cells is mediated by a structurally and functionally unique ACh receptor (AChR). We propose to study the cholinergic response of hair cells isolated from the cochlea of the chick in an effort to understand the cellular and molecular bases of this inhibition. In addition, we intend to study the developmental acquisition of sensitivity to ACh in order to learn what factors govern the selective expression of these receptors in outer hair cells in adults. The experiments to meet these goals will be largely electrophysiological in nature, reflecting the established expertise of this laboratory. These will include whole-cell, cell-attached and excised patch voltage-clamp recordings. Also, we will use fluorescence microscopy of intracellular signals from Ca-indicator dyes in order to assess the role of Ca as a second messenger in the hair cell response to ACh. The initial goal of our experiments in molecular biology will be to identify the genes coding for the subunits of the hair cell AChR. We will first use low-stringency homology screening of a chick's cochlear cDNA library to identify transcripts with some relatedness to known AChRs. Expression of identified clones in hair cells will be examined by in situ hybridization and potentially by reverse transcriptase polymerase chain reaction amplification. Functional reconstitution studies will be performed by injection of cRNA into Xenopus oocytes. The developmental acquisition of AChRs by hair cells will be studied during the last 10 days of embryogenesis, that is, preceding and spanning the time that efferent axons make calyciform synapses in the cochlea. Initially we will ask whether there are parallel changes in the appearance of identified mRNAs and hair cell sensitivity to ACh. Antisense RNA will be used to block expression of identified gene products. The motivation for this study is multi-part: to understand better the mechanisms of cochlear inhibition, to contribute to the study of the molecular biology and biophysics of AChRs, and to begin to study the factors governing the developmental maturation of hair cell sensitivity to ACh.
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