Improved preservation of the otolithic membranes

Abstract

Any explanation of vestibular micromechanics must include the roles of the otolithic and cupular membranes. However, micromechanical models of vestibular function have been hampered by unresolved questions about the microarchitectures of these membranes and their connections to stereocilia and supporting cells. Otolithic membranes are notoriously difficult to preserve because of severe shrinkage and loss of soluble components. We have empirically developed fixation procedures that reduce shrinkage artifacts and more accurately depict the spatial relations between the otolithic membranes and the ciliary bundles and supporting cells.We used White Leghorn chicks, ranging in age from newly hatched to one week. The inner ears were fixed for 3-24 h in 1.5-1.75% glutaraldehyde in 150 mM KCl, buffered with potassium phosphate, pH 7.3; when postfixed, it was for 30 min in 1% OsO4 alone or mixed with 1% K4Fe(CN)6. The otolithic organs (saccule, utricle, lagenar macula) were embedded in Araldite 502. Semithin sections (1 μ) were stained with toluidine blue.