Abstract
BACKGROUND: Intense endocytic activity at the apex of outer hair cells (OHCs)-the electromechanical cells of the cochlea-has been demonstrated using the vital plasma-membrane marker FM1-43 and confocal laser-scanning microscopy. Vesicular traffic toward the cell nucleus to distinct locations of the endoplasmic reticulum has also been shown. OBJECTIVE: The current study characterizes the dynamics of endocytic activity, as well as apicobasal and basoapical trafficking, using a local perfusion technique that we recently developed and published to visualize bidirectional trafficking in isolated bipolar cells. MATERIALS AND METHODS: The fluorescent plasma-membrane markers FM1-43 (10 µM) and FM4-64 (10 µM), together with a fluid-phase marker, Lucifer yellow (50 µM), were used to label endocytosed vesicles in isolated OHCs of the guinea pig cochlea. Targets of endocytosed vesicles were examined with a fluorescent marker of subsurface cisternae, DiOC(6) (0.87 µM). Single- and two-photon confocal laser-scanning microscopy was used to visualize labeled vesicles. RESULTS: The plasma-membrane markers presented more intense vesicle internalization at the synaptic pole than at the apical pole of the OHC. Intracellular basoapical vesicle trafficking was faster than apicobasal trafficking. Vesicles endocytosed at the synaptic pole were transcytosed to the endoplasmic reticulum system. An intracellular Lucifer yellow signal was not detected. CONCLUSION: The larger endocytic fluorescent signals in the synaptic pole and the faster basoapical trafficking imply that membrane internalization and vesicle trafficking are more efficient at the synaptic pole than at the apical pole of the OHC.