3-D ANALYSIS OF F-ACTIN IN STEREOCILIA OF COCHLEAR HAIR-CELLS AFTER LOUD NOISE EXPOSURE

Fluorescence microscopy can be a useful tool in the early detection of pathological changes in the stereocilia of outer hair cells which hav e undergone acoustic overstimulation. Fluorescent phalloidin, a highly specific F-actin stain, can be used to label F-actin in stereocilia. In this study, phalloidin label is used to determine quantitative chan ges of F-actin in the stereocilia of guinea pigs exposed to loud noise (117 dB; octave band noise, centered at 1 kHz; 4 h). Reliably determi ning three-dimensional (3-D) structural changes in stereocilia is a ch allenging problem in optical microscopy since stereocilia diameter is close to the optical resolution limit. In order to alleviate the probl em, a computational 3-D microscopy technique is used (Avinash et al., 1992). Whole-mounts of the cochlear second and third turns were examin ed in a Leitz Orthoplan microscope through a Leitz Plan Apo objective lens (100 X; 1.32 N.A.; 170/0.17). Images were acquired with a charge- coupled device camera where the focus was shifted in 0.2 mum steps usi ng a piezoelectric translator. Images were processed with the appropri ate point spread function of the optical system. Analysis of control c ochleas indicate that our technique can resolve single stereocilia and distinguish between various intensities of label along each stereocil ia. In noise-exposed cochleas, our data show length and intensity chan ges in the phalloidin label. These results suggest that both depolymer ization and polymerization of F-actin can occur in stereocilia of oute r hair cells after acoustic overstimulation. Our findings demonstrate the applicability of computational 3-D microscopy to quantitative and qualitative analysis of stereocilia.