OBJECTIVE: To investigate the expression of adaptin-2(AP-2) in mice cochlea and to discuss the probable role in the endocytosis of hair cells. METHOD: Laser scanning confocal microscopy and immune-fluroscence histochemistry were performed in this study. RESULT: In mature mice cochlea, the immunoreactivity for AP-2 was found in the inner hair cells cytoplasm. This protein mainly expressed in the hair cells basal part and nearby the ribbon synapse. CONCLUSION AP-2 protein mainly expressed in the hair cells synaptic activity zone , which suggested that AP-2 could play an important role in the synaptic vesicle endocytosis. This finding built the foundation for the further research involved in the physiological and pathological role of AP-2.
Adaptor Protein Complex alpha Subunits ; metabolism ; Animals ; Cochlea ; Hair Cells, Auditory ; Hair Cells, Auditory, Inner ; metabolism ; Mice ; Microscopy, Confocal ; Synapses

OBJECTIVE: To investigate the expression of Myosin VI and Disabled-2 (Dab2) in the cochlea of mice at different ages. METHOD: Forty KM mice were divided into four groups according to age, named as postnatal 2 week (P2w), P5w, P9w, P16month. The localization of protein in the basilar membrane of mice cochlea was detected by immunofluorescence staining and laser scanning confocal microscope (LSCM). The mRNA expression level of protein in cochlear at different ages was evaluated by real-time fluorescent quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Statistical analysis was performed by the SPSS18.0 software. RESULT: Myosin VI and Disabled-2 protein mainly expressed at the apical cytoplasm of hair cells. As for the inner hair cell, Dab2 labeling was abundant especially at the cuticular plate and nearby. Comparing four immunofluorescence staining images of Myosin VI, we found the fluorescence intensity of P2w and P16m were weaker than that of P5w and P9w. After setting P9w as the control group, qRT-PCR revealed that the mRNA expression of MyosinVI and Dab2 in P2w was less than that in the control group (P < 0.01), while no significant difference was found between P5w and the control group, nor between P16m and the control group (P > 0.05). CONCLUSION Myosin VI and Dab2, two proteins which regulated the clathrin-mediated endocytosis, expressed at hair cells of mice cochlea. In the inner hair cell, this process of endocytosis may be more efficient at the cuticular plate and nearby. The expression level of protein may change in different ages, and this probably leads to a difference of CME, it also may cause a defect of inner hair cells function.
Adaptor Proteins, Vesicular Transport ; metabolism ; Aging ; Animals ; Cochlea ; metabolism ; Endocytosis ; Hair Cells, Auditory ; metabolism ; Hair Cells, Auditory, Inner ; metabolism ; Mice ; Microscopy, Confocal ; Myosin Heavy Chains ; metabolism

The cochlear auditory epithelium contains two types of sound receptors, inner hair cells (IHCs) and outer hair cells (OHCs). Mouse models for labelling juvenile and adult IHCs or OHCs exist; however, labelling for embryonic and perinatal IHCs or OHCs are lacking. Here, we generated a new knock-in Fgf8^P2A-3×GFP/+ (Fgf8^GFP/+) strain, in which the expression of a series of three GFP fragments is controlled by endogenous Fgf8 cis-regulatory elements. After confirming that GFP expression accurately reflects the expression of Fgf8, we successfully obtained both embryonic and neonatal IHCs with high purity, highlighting the power of Fgf8^GFP/+. Furthermore, our fate-mapping analysis revealed, unexpectedly, that IHCs are also derived from inner ear progenitors expressing Insm1, which is currently regarded as an OHC marker. Thus, besides serving as a highly favorable tool for sorting early IHCs, Fgf8^GFP/+ will facilitate the isolation of pure early OHCs by excluding IHCs from the entire hair cell pool.
Animals ; Mice ; Hair Cells, Auditory, Inner ; Cochlea/metabolism* ; Hair Cells, Auditory, Outer/metabolism* ; Disease Models, Animal ; Fibroblast Growth Factor 8/metabolism*

Recent studies have revealed great functional and structural heterogeneity in the ribbon-type synapses at the basolateral pole of the isopotential inner hair cell (IHC). This feature is believed to be critical for audition over a wide dynamic range, but whether the spatial gradient of ribbon morphology is fine-tuned in each IHC and how the mitochondrial network is organized to meet local energy demands of synaptic transmission remain unclear. By means of three-dimensional electron microscopy and artificial intelligence-based algorithms, we demonstrated the cell-wide structural quantification of ribbons and mitochondria in mature mid-cochlear IHCs of mice. We found that adjacent IHCs in staggered pairs differ substantially in cell body shape and ribbon morphology gradient as well as mitochondrial organization. Moreover, our analysis argues for a location-specific arrangement of correlated ribbon and mitochondrial function at the basolateral IHC pole.
Animals ; Artificial Intelligence ; Cochlea/metabolism* ; Hair Cells, Auditory, Inner ; Mice ; Mitochondria ; Synapses/metabolism*

In order to elucidate the mechanism underlying the attenuation of streptomycin ototoxicity by tetramethylpyrazine (TMP), the present study investigated the effect of TMP on the outward K(+) current in the outer hair cells of guinea pig cochlea. Sixty guinea pigs were divided into 6 groups randomly. Auditory brainstem response (ABR) was used to observe the change in thresholds and to evaluate ototoxicity induced by streptomycin. Whole-cell patch-clamp technique was used to observe the effect of TMP on outward K(+) current in isolated outer hair cells. The results showed that TMP attenuated the threshold shift caused by streptomycin and increased the amplitudes of Ca(2+)-sensitive K(+) current [I(K(Ca))] in the outer hair cells. The present data suggest that TMP displays anti-ototoxicity induced by streptomycin. The augmented amplitudes of I(K(Ca)) of the outer hair cells induced by TMP may be one of the mechanisms underlying its ototoxicity-attenuating effect.
Animals ; Auditory Threshold ; Cochlea ; cytology ; Evoked Potentials, Auditory, Brain Stem ; Guinea Pigs ; Hair Cells, Auditory, Outer ; drug effects ; Patch-Clamp Techniques ; Potassium Channels ; metabolism ; Pyrazines ; Streptomycin ; toxicity

In mammals, the piezoelectric protein, Prestin, endows the outer hair cells (OHCs) with electromotility (eM), which confers the capacity to change cellular length in response to alterations in membrane potential. Together with basilar membrane resonance and possible stereociliary motility, Prestin-based OHC eM lays the foundation for enhancing cochlear sensitivity and frequency selectivity. However, it remains debatable whether Prestin contributes to ultrahigh-frequency hearing due to the intrinsic nature of the cell's low-pass features. The low-pass property of mouse OHC eM is based on the finding that eM magnitude dissipates within the frequency bandwidth of human speech. In this study, we examined the role of Prestin in sensing broad-range frequencies (4-80 kHz) in mice that use ultrasonic hearing and vocalization (to >100 kHz) for social communication. The audiometric measurements in mice showed that ablation of Prestin did not abolish hearing at frequencies >40 kHz. Acoustic associative behavior tests confirmed that Prestin-knockout mice can learn ultrahigh-frequency sound-coupled tasks, similar to control mice. Ex vivo cochlear Ca²⁺ imaging experiments demonstrated that without Prestin, the OHCs still exhibit ultrahigh-frequency transduction, which in contrast, can be abolished by a universal cation channel blocker, Gadolinium. In vivo salicylate treatment disrupts hearing at frequencies <40 kHz but not ultrahigh-frequency hearing. By pharmacogenetic manipulation, we showed that specific ablation of the OHCs largely abolished hearing at frequencies >40 kHz. These findings demonstrate that cochlear OHCs are the target cells that support ultrahigh-frequency transduction, which does not require Prestin.
Animals ; Cochlea/metabolism* ; Hair Cells, Auditory, Outer/metabolism* ; Hearing ; Humans ; Mammals/metabolism* ; Mice ; Mice, Knockout ; Molecular Motor Proteins/metabolism*

OBJECTIVE: To detect the expression variation of microRNA-183 family in cochlea of animal model characterized by noise-induced deafness at various time points, and to explore the mechanisms responsible for noise-induced deafness. METHOD: Fifty mice were randomly divided into 5 groups. In the experimental group, 40 mice were exposed to 2-4 kHz narrow band noise at 100 dB SPL 6h per day for 3 consecutive days. The rest 10 mice served as the control group without receiving any noise. Auditory brainsterm response (ABR) were examined at the 1st, 7th, 14th and 28th day compaired with the ABR before the experiment,to confirm noise lead to the permanent threshold shift. The pathological damage processes of hair cell were detected by the basilar membrane stretched techniques. Real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was apply to quantify the expression of microRNA183 family members. Statistical analysis was performed by the SPSS 17.0 software. RESULT: The hearing of mice in the experimental group was significantly less than that in the control group. In the experimental group, the hearing of mice exposed to noise were markedly less when compared with the one exposure to null-noise. The hearing in the 1st day group was least among experimental groups, and the followed one was mice in the 7th day group. No statistical difference were observed between the 14th and 28th day groups (P > 0.05). The results of surface preparation showed that the outer hair cells were chaotic, deformational, and their number decreased is time-dependent. The missing of the outer hair cells occurred mainly in the first and second rows, while the inner hair cells were not pronouncedly missing. The qRT-PCR showed that the expressions of the three genes (miR-183/96/182)in the 1st day and 7th day group with exposure to noise were less than in the control group (P < 0.01), while no significant difference was found between 1st day and 7th day group (P > 0.05). The expressions rised in the 14th day experimental groups, whereas the 28th day group's expressions of the three genes decreased markedly which were more than that in the 1st day and 7th day group (P < 0.01). CONCLUSION After noise exposure for some time, the expressions of miRNA-183 family members have significant changes in animal model with noise-induced deafness, which indicated that the miRNA183 family members may play important roles in the pathogenesis and development of noise-induced deafness.
Animals ; Disease Models, Animal ; Evoked Potentials, Auditory, Brain Stem ; Female ; Hair Cells, Auditory, Inner ; pathology ; Hair Cells, Auditory, Outer ; pathology ; Hearing Loss, Noise-Induced ; metabolism ; pathology ; Male ; Mice ; MicroRNAs ; metabolism

OBJECTIVETo investigate the pathway and mechanism of noise exposure induced out hair cells (OHC) apoptosis.

METHODSThe cochleae of control and noise exposure group were dissected. The activity of caspase 3, an important mediator of apoptosis, in OHC, was examined with carboxyfluorescein-labeled fluoromethyl ketone (FMK)-peptide inhibitors. The apoptosis inducing factor (AIF) translocation from mitochondria in OHC were further examined by immunohistology method. The nuclei were labeled with PI and the mitochondrion was labeled with Mito-tracker. Whole mount organ of Corti was prepared. Morphological and fluorescent change was observed use confocal microscope.

RESULTSIn the normal OHC, AIF is distributed where the mitochondria were located and no activated caspase 3 was observed. After the animals exposed to broadband noise at 122 dB in 4 h/day for 2 days, both apoptosis and necrosis were appeared in OHC. AIF translocated from mitochondrion to nuclei in apoptotic and necrotic OHC following noise exposure. The noise exposure triggered activation of caspase 3 in apoptic hair cells. But no caspase 3 activation appeared in necrotic OHC.

CONCLUSIONSThese findings indicated that the caspase-dependent pathway is an important pathway in noise exposure induced apoptosis. And AIF also involves OHC death pathway following noise exposure.

Animals ; Apoptosis ; Apoptosis Inducing Factor ; metabolism ; Caspase 3 ; metabolism ; Female ; Guinea Pigs ; Hair Cells, Auditory, Outer ; metabolism ; pathology ; Male ; Noise

OBJECTIVE: To learn the influence the gentamycin on C57BL/6J mice hear and cochlear hair cell ribbon synapses CaV1.3 calcium protein amount. To explore the relationship between hear loss and its dosage correlation change and significance. METHOD: The fixed amino glucoside to C57BL/6J mice was used to make abdominal cavity injection mold every day. The auditory brain-stem response ABR was used to measure the hear of mice in 7th, 14th, 28th after the injection. Immunofluorescence method was used to observe cochlear basement membrane of hair ribbon synapse CaV1.3 calcium channel proteins in the distribution and expression. Inner hair cells synaptic membrane was immune fluorescent tags with CtbP2 and CaV1. 3. RESULT: With the growth of the injected drugs, ABR threshold increased,but all the hair cells and shape had no obvious change. However the amount of hair rib bon synapse CaV1.3 calcium ion channel proteins in the expression had significant differences (P < 0.01). CaV1.3 calcium ion channel proteins increased slightly lower than normal at 7th day, significantly decreased at 14th day, had increased, increased quantity compare with 14th day, but at 28th day after intraperitoneal injection of gentamicin. CONCLUSION The increasing,decreasing and increasing trend of cochlear hair cells CaV1.3 proteins in the environment of amino glucoside drug toxicity showed that the increase of hair ribbon synapse CaV1.3 proteins may have a compensatory effect on the drug toxicity. With the increase of the drug toxicity effect, this kind of decompensated function could be the listening decline, which may be one of the mechanism of damage to hearing.
Animals ; Calcium Channels, L-Type ; metabolism ; Evoked Potentials, Auditory, Brain Stem ; Gentamicins ; pharmacology ; Hair Cells, Auditory, Inner ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Proteomics

OBJECTIVETo explore the protective effects of earplug and barrel on auditory organs of guinea pigs exposed to experimental blast underpressure (BUP).

METHODSThe hearing thresholds of the guinea pigs were assessed with auditory brainstem responses (ABR). The traumatic levels of tympanic membrane and ossicular chain were observed under stereo-microscope. The rate of outer hair cells (OHCs) loss was analyzed using a light microscope. The changes of guinea pigs protected with barrel and earplug were compared with those of the control group without any protection.

RESULTSAn important ABR threshold shift of the guinea pigs without any protection was detected from 8h to 14d after being exposed to BUP with a peak ranging from -64.5 kPa to -69.3 kPa ( P<0.01). The rate of perforation of tympanic membrane reached 87.5% and that of total OHCs loss was 19.46% +/- 5.38% at 14d after exposure. The guinea pigs protected with barrel and earplug had lower ABR threshold and total OHCs loss rate compared with the animals without any protection (P<0.01). All of the tympanic membrane and ossicular chain of the protected animals maintained their integrities. Meanwhile, the guinea pigs protected with the barrel had lower ABR threshold and total OHCs loss rate than those with earplug (P<0.01).

CONCLUSIONSThe earplug and barrel have protective effects against BUP-induced trauma on auditory organs of the guinea pigs and the protective effects of barrel are better than those of earplug.

Animals ; Auditory Threshold ; Blast Injuries ; prevention & control ; Ear Protective Devices ; Guinea Pigs ; Hair Cells, Auditory, Outer ; metabolism ; Pressure ; Tympanic Membrane ; injuries ; Tympanic Membrane Perforation ; etiology ; physiopathology ; prevention & control