Reactive oxygen species (ROS) have been suggested to play a major role in aminoglycoside-induced hair cell (HC) loss, but are difficult to detect. Moreover, ROS can occur normally in cells where they have roles in metabolism, cell signaling and other processes. Two new probes, aminophenyl fluorescein (APF) and hydroxyphenyl fluorescein (HPF) are dyes which selectively detect highly-reactive oxygen species (hROS), those most associated with cellular damage. We assessed the presence of hROS in the neonatal rat organ of Corti during chronic exposure to 50 μM gentamicin in vitro, to examine the relationship between cell damage and hROS across HC type and across the three cochlear turns. hROS were initially detected at 48 hours (h), with an increase at 72 h and persistence until at least 96 h. At 48 h, hROS were restricted to outer HCs and occurred prior to loss of stereocilia. At 72 h, outer HCs showed both hROS and stereocilia loss, and hROS were noted in a few inner HCs. Basal turn HCs showed more hROS than middle turn HCs. Very little hROS accumulation or stereocilia loss was observed in the apical turn, even at 72 h. First row outer HCs were most vulnerable to gentamicin-induced hROS, followed by second and then third row outer HCs. Inner HCs behaved similarly to third row outer HCs. By 96 h stereocilia damage was extensive, but surviving HCs showed persisting fluorescence. APF consistently showed more fluorescence than HPF. The results suggest that hROS accumulation is an important initial step in gentamicin-induced HC damage, and that the differential sensitivity of HCs in the organ of Corti is closely related to differences in hROS accumulation.

Olfactory receptor neurons (ORNs) use odour-induced intracellular cAMP surge to gate cyclic nucleotide-gated nonselective cation (CNG) channels in cilia. Prolonged exposure to cAMP causes calmodulin-dependent feedback-adaptation of CNG channels and attenuates neural responses. On the other hand, the odour-source searching behaviour requires ORNs to be sensitive to odours when approaching targets. How ORNs accommodate these conflicting aspects of cAMP responses remains unknown. Here, we discover that olfactory marker protein (OMP) is a major cAMP buffer that maintains the sensitivity of ORNs. Upon the application of sensory stimuli, OMP directly captured and swiftly reduced freely available cAMP, which transiently uncoupled downstream CNG channel activity and prevented persistent depolarization. Under repetitive stimulation, OMP-/-ORNs were immediately silenced after burst firing due to sustained depolarization and inactivated firing machinery. Consequently, OMP-/mice showed serious impairment in odour-source searching tasks. Therefore, cAMP buffering by OMP maintains the resilient firing of ORNs.

Here, we report the functional and morphological evidence of hair cell recovery after damages induced by gentamicin (GM) in cultured explants of rat vestibular maculae. We evaluated mechano-electrical transduction (MET) function in hair cells, by measuring Ca(2+) responses in the explants with fura-2 when hair bundles were stimulated. After the MET testing, hair bundles were observed in high resolution by scanning electron microscopy, or by fluorescence microscopy after staining with phalloidin-FITC (fluorescent isothiocyanate). In the control culture, the number of hair bundles on the explants gradually decreased, and the percentage of explants showing Ca(2+) responses decreased and disappeared after 17 days in culture. Following GM (1-2 mM) treatment, most of the hair bundles were eliminated initially, but the hair bundles gradually increased in number during culture. Short hair bundle-like structures emerged in the areas where hair bundles had been completely lost. Consistent with the morphological observations, Ca(2+) responses disappeared after GM treatment, and they gradually recovered to a peak 13-17 days after treatment and were even induced at 17 days or more in culture. Furthermore, cells accumulated FM1-43, a dye permeable through the MET channel, when Ca(2+) responses recovered after GM treatment. Application of steroid hormone increased the percentage of explants showing MET activity, and enhanced the recovery of MET after GM treatment. We investigated Ki-67 immunoreactivity to detect cell proliferation and TUNEL staining to detect apoptotic cell death. Ki-67 immunoreactivity was negative after GM treatment, however TUNEL staining was positive and the positivity was GM dose dependent. Therefore, this functional recovery of transduction activity was not owing to the proliferation of hair cells but was likely the self-repair of the hair bundle.

Once inner ear hair cells (HCs) are damaged by drugs, noise or aging, their apical structures including the stereociliary arrays are frequently the first cellular feature to be lost. While this can be followed by progressive loss of HC somata, a significant number of HC bodies often remain even after stereociliary loss. However, in the absence of stereocilia they are nonfunctional. HCs can sometimes be regenerated by Atoh1 transduction or Notch inhibition, but they also may lack stereociliary bundles. It is therefore important to develop methods for the regeneration of stereocilia, in order to achieve HC functional recovery. Espin is an actin bundling protein known to participate in sterociliary elongation during development. We evaluated stereociliary array regeneration in damaged vestibular sensory epithelia in tissue culture, using viral vector transduction of two espin isoforms. Utricular HCs were damaged with aminoglycosides. The utricles were then treated with a γ-secretase inhibitor, followed by espin or control transduction and histochemistry. While γ-secretase inhibition increased the number of HCs, few had stereociliary arrays. In contrast, 46 hrs after espin1 transduction, a significant increase in hair-bundle-like structures was observed. These were confirmed to be immature stereociliary arrays by scanning electron microscopy. Increased uptake of FM1–43 uptake provided evidence of stereociliary function. Espin4 transduction had no effect. The results demonstrate that espin1 gene therapy can restore stereocilia on damaged or regenerated HCs.