LAUSR

Intratympanic injection of gentamicin has proven to be an effective therapy for intractable vestibular dysfunction. However, most studies to date have focused on the cochlea, so little is known about the distribution and uptake of gentamicin by the counterpart of the auditory system, specifically vestibular hair cells (HCs). Here, with a combination of in vivo and in vitro approaches, we used a gentamicin‐Texas Red (GTTR) conjugate to investigate the mechanisms of gentamicin vestibulotoxicity in the developing mammalian utricular HCs. In vivo, GTTR fluorescence was concentrated in the apical cytoplasm and the cellular membrane of neonatal utricular HCs, but scarce in the nucleus of HCs and supporting cells. Quantitative analysis showed the GTTR uptake by striolar HCs was significantly higher than that in the extrastriola. In addition, the GTTR fluorescence intensity in the striola was increased gradually from 1 to 8 days, peaking at 8–9 days postnatally. In vitro, utricle explants were incubated with GTTR and candidate uptake conduits, including mechanotransduction (MET) channels and endocytosis in the HC, were inhibited separately. GTTR uptake by HCs could be inhibited by quinine, a blocker of MET channels, under both normal and stressed conditions. Meanwhile, endocytic inhibition only reduced GTTR uptake in the CoCl2 hypoxia model. In sum, the maturation of MET channels mediated uptake of GTTR into vestibular HCs. Under stressed conditions, MET channels play a pronounced role, manifested by channel‐dependent stress enhanced GTTR permeation, while endocytosis participates in GTTR entry in a more selective manner.