The goal of the proposed study is to examine the expression of the three known alpha-bungarotoxin (alpha-BTX) sensitive subunits, alpha 7, alpha 8, and alpha 9, of the nicotinic acetylcholine receptor. The degenerate polymerize chain reaction (PCR) cloning technique will be used to clone probes for three nicotinic acetylcholine receptor subunits in the guinea pig cochlea. DNA sequencing will be performed on these cloned fragments to determine their nucleotide sequence, thus, establishing their identity as one of the known receptor subunits or perhaps a new auditory-specific form of an alpha-BTX sensitive nicotinic ACh receptor. The second specific aim is to determine the cellular expression pattern of these nicotinic ACh receptor subunits in the guinea pig cochlea. This will be accomplished by in situ hybridization techniques using a colorimetric detection protocol, rather than radioisotopic detection, to provide cellular resolution. Additionally, co-localization studies are proposed using riboprobes labeled with different fluorophores in conjunction with confocal microscopy.
| Hiel, H; Luebke, A E; Fuchs, P A (2000) Cloning and expression of the alpha9 nicotinic acetylcholine receptor subunit in cochlear hair cells of the chick. Brain Res 858:215-25 |
