FM1-43 reveals membrane recycling in adult inner hair cells of the mammalian cochlea

Neural transmission of complex sounds demands fast and sustained rates of synaptic release from the primary cochlear receptors, the inner hair cells (IHCs). The cells therefore require efficient membrane recycling. Using two-photon imaging of the membrane marker FM1-43 in the intact sensory epithelium within the cochlear bone of the adult guinea pig, we show that IHCs possess fast calcium-dependent membrane uptake at their apical pole. FM1-43 did not permeate through the stereocilial mechanotransducer channel because uptake kinetics were neither changed by the blockers dihydrostreptomycin and D-tubocurarine nor by treatment of the apical membrane with BAPTA, known to disrupt mechanotransduction. Moreover, the fluid phase marker Lucifer Yellow produced a similar labeling pattern to FM1-43, consistent with FM1-43 uptake via endocytosis. We estimate the membrane retrieval rate at similar to0.5% of the surface area of the cell per second. Labeled membrane was rapidly transported to the base of IHCs by kinesin-dependent trafficking and accumulated in structures that resembled synaptic release sites. Using confocal imaging of FM1-43 in excised strips of the organ of Corti, we show that the time constants of fluorescence decay at the basolateral pole of IHCs and apical endocytosis were increased after depolarization of IHCs with 40 mM potassium, a stimulus that triggers calcium influx and increases synaptic release. Blocking calcium channels with either cadmium or nimodipine during depolarization abolished the rate increase of apical endocytosis. We suggest that IHCs use fast calcium-dependent apical endocytosis for activity-associated replenishment of synaptic membrane.