Morphological and physiological development of vestibular hair cells in the organ-cultured otocyst of the chick.

The inner ear of the embryonic chick forms an oval-shaped sac or otocyst, on Embryonic Day 3, which contains presumptive sensory and support cells. After 3 weeks in organ culture the otocyst had sensory epithelia with an average of 325 +/- 41 hair cells. Using light and transmission electron microscopy most of these cells were identified morphologically as type II vestibular hair cells. Whole-cell tight-seal recordings, using potassium chloride-filled micropipetes, showed that mature cultured hair cells had four different types of K+ currents. These included: a voltage-gated delayed rectifier K+ current (IK), an inactivating K+ current (IA), a calcium-dependent K+ current (IK(Ca)), and a K+ inward rectifier (IIR). These currents were similar to those recorded from cristae ampullares cells isolated from 2- to 3-week-old posthatched chicks. We also determined the timing of K+ current acquisition in vitro. Initially, recordings showed that cells isolated from Embryonic Day 3 otocysts had no voltage-dependent outward currents at physiological membrane potentials. Eventually, K+ currents were acquired in the order of: IK and IIR after 9 days, IA after 12 days, and IK(Ca) after 17 days in vitro. In addition, recordings using cesium chloride-filled micropipetes showed that there were two types of inward currents that were elicited in response to membrane depolarizations. These two currents included a rapidly activating, noninactivating Ca2+ current and a tetrodotoxin-sensitive Na+ current. Both currents were elicited in hair cells grown in vitro for 13 days. Although INa was previously unreported in avians, both INa and ICa were also represented in hair cells isolated from the cristae ampullares of the posthatched chick. These results indicate that hair cells can acquire voltage-gated currents in vitro which are qualitatively similar to ionic currents found in crista ampullaris cells that differentiate in vivo. Thus, this organ culture system provides a means to study regulation of ionic currents in developing hair cells.