Our long term goal is to understand how cochlear hair cells function as an effective frequency analyzer of the peripheral auditory system. The goals of this proposal are to identify members of the voltage-gated ion channel superfamily expressed in cochlear hair cells and to determine their spatial and temporal patterns of expression in developmental and adult stages. These studies will seek to correlate the anatomical maturation and neuronal innervation of the organ of Corti with the functional maturation of the cochlear hair cells' receptor potential and synaptic transmission. Our studies will focus on characterization of the ion channels that shape the receptor potential at the molecular level. These channels include the voltage-gated K+ (Kv) and Na+ (Scn) ion channels and the inward rectifiers (Kir). The voltage-dependent Ca2+ (CACN) channels and calcium-activated K+ channels (Kca), which are involved in cochlear hair cell synaptic transmissions, will also be characterized. Initial gene expression analysis of these ion channels will use degenerative gene-family and gene-specific oligonucleotide primers to amplify cDNAs from cochlear, inner hair cell, and outer hair cell cDNA libraries as well as single cell RT-PCR.
The first aim will be to characterize the hair cells' ion channels in normal adult rats and mice at the molecular level using wholemount and section preparations of the organ of Corti and applying probes directed at candidate proteins (immunocytochemistry) and mRNA (in situ hybridization). Once the adult spatial expression patterns are established, we will study the spatio-temporal expression patterns in mice during a prenatal and neonatal time course to determine acquisition time of these ion channels and to correlate these changes with innervation and anatomical maturation. Lastly we will determine the spatial pattern of ion channel expression in null mutant mice (Ngn-1, Ntrk2 (trkB), Ntf3 (Nt-3), Ntrk2+/Ntf3+/-, and Ntf3+-PDGF promoter-Ntf3+) to elucidate the relationship between innervation and electrophysiological maturation. Identification of molecular elements involved in the receptor potential and synaptic transmission of cochlear hair cells should lead to a greater understanding of inner ear function and contribute to our comprehension of auditory pathologies.
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