Iwashita et al. 1999; Wysocki and Ladich 2001) or Fish were prepared for microscopic analysis by found no change between different sizes of fish (Pop- euthanizing with MS-222 (an anesthetic for coldper 1971). However, only the elasmobranch study blooded vertebrates). The total length (tip of snout (Corwin 1983) directly correlated the responses of the to tip of tail) was determined for each animal used in ear with changes in the number of sensory hair cells. this study. The jaw and tissue covering the otic capsules Zebrafish have become a particularly important were then removed and the remaining tissue, conmodel for vertebrate genetic studies, and there is an taining the ear, was placed in 2.0% paraformaldehyde increasing number of studies on the genetics and and used as described below. development of the ear in this species (Granato et al. Phalloidin and hair cell counts. The number of saccular 1996; Riley and Grunwald 1996; Whitfield et al. 1996; hair cells was determined for uncrowded zebrafish Riley and Moorman 2000). In spite of this importance, aged 3, 6, 10, 15, and 18 months and for crowded there have been few studies on the zebrafish auditory zebrafish aged 10 and 27 months. The saccule is system (Waterman and Bell 1984; Platt 1993; Haddon thought to be the primary auditory endorgan in this and Lewis 1996) and no studies examining zebrafish group (reviewed in Platt and Popper 1981; Fay 1988b), hearing ability. The purpose of the current study was so only saccular hair cells were considered here. At to examine developmental changes in the number of least three fish were used for each age group, with up sensory hair cells in the saccule of zebrafish (Danio to six for the youngest animals where more specimens rerio) aged three months to over two years, represent- were available. Fish were sacrificed and the tissue fixed ing animals from sexual immaturity to adulthood. We in 2% paraformaldehyde for 30 minutes. The saccules also examined the hearing ability of zebrafish to inves- were then removed from the heads, rinsed in phostigate possible relationships between hair cell number phate buffer, and placed in Oregon Green Phalloidin and hearing ability. In addition, we wanted to test the (Molecular Probes, Inc., Eugene, OR) for 30 minutes. hypothesis that hair cell addition slows or stops when Phalloidin selectively binds to the actin in hair cell overall growth of the fish stops. This hypothesis is parbundles, making them easy to identify. The tissue was ticularly important if zebrafish become models for rinsed in phosphate buffer, mounted using Prolong genetic effects on adult or aging ears since mainte-Antifade (Molecular Probes, Inc.), and examined nance of fish under conditions where growth is artifiusing a Biorad MC 1024 confocal microscope. Low cially slowed (eg, crowded conditions) could have power images (40X objective) of the saccular epitheprofound effects on interpretation of results. lium were taken and then manually merged to get a