A new method was developed for specific labeling of vestibular afferent and efferent neurons from selected end-organs. Biocytin solution was applied to the inner ear after the surface of the sensory neuroepithelium of an end-organ had been scratched. After 24 hours, the animal was perfused with a fixative and its brainstem was removed. The brainstem and vestibular nerves were serially sectioned with a freezing microtome, and 40 μm slices were processed with ABC solution. Diaminobenzidine (DAB) was used as the chromagen. Sections were mounted on slides and coverslipped for light microscopic observation. The distribution of primary afferent fibers from the horizontal semicircular canal (HSC) and the saccule (SAC) in the brainstem was as previously reported (Lee WS, 1992). The selectivity of efferent labeling was verified by the distribution of afferent fibers labeled simultaneously. A group of vestibular efferent neurons was found in the dorsal area of the brainstem ventromedial to the medial vestibular nucleus and lateral to the facial genu. Another group of efferents was situated ventrally in the reticular formation. Dorsal group efferent neurons innervating HSC were located more caudally than SAC efferents and were primarily on the contralateral side. In the reticular formation, SAC efferents were found more on the contralateral side, while HSC efferents were found bilaterally. This method may contribute to the detailed anatomical study of the vestibular efferent system.
