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*

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*

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

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

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 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

OBJECTIVETo explore the feature of the ACh-sensitive potassium current in guinea pig cochlear outer hair cells.

METHODSCochlear outer hair cells of guinea pigs (n=38) were isolated by collagenase type IV. Under the whole-cell patch mode, the ions nature and the pharmacological properties of the ACh-sensitive potassium current were investigated by applying the inhibitors of calcium-dependent potassium currents and the inhibitors of nicotinic ACh receptor.

RESULTSFollowing application of ACh, cochlear outer hair cells displayed a rapidly activating outward potassium current with a fast desensitized kinetic and a reversal (x +/- s) potential of (-67.3 +/- 8.2) mV (n=10). At the holding potential of -50 mV, the current amplitude of ACh-sensitive potassium current activated by 100 micronmol/L ACh was (506.6 +/- 186.3) pA (n=9). ACh-sensitive potassium current was sensitive to TEA (tetraethylammonium chloride, 10 mmol/L) and potently inhibited by the small conductance calcium-dependent potassium current (SK) blocker, apamin (1 micromol/L). Iberiotoxin (IBTX), the well-known blocker of big conductance calcium-dependent potassium current (BK), failed to inhibit the amplitude of the ACh-sensitive potassium current at the dose of 200 nmol/L. The dose for half-maximal response (EC50) of the ACh-sensitive potassium current was (33.5 +/- 5.7) micromol/L (n=7). The ACh-sensitive potassium current was sensitive to the GABA (gamma-aminobutyric acid)-A receptor blocker, bicuculline, and strongly inhibited by the selective blocker of the alpha 9-nicotinic ACh receptor, strychnine. Strychnine and bicuculline showed the dose-dependent blocking effect with a half inhibition-maximal response (IC50) of (22.3 +/- 2.6) nmol/L (n=7) and (1.2 +/- 0.4) micromol/L (n=6), respectively.

CONCLUSIONSThis work provides direct evidences that the ACh-sensitive SK current was present on guinea pig cochlear outer hair cells. The activation of the ACh-sensitive SK current was most possibly mediated by a alpha 9-nicotinic ACh receptor.

Animals ; Guinea Pigs ; Hair Cells, Auditory, Outer ; drug effects ; metabolism ; Membrane Potentials ; Patch-Clamp Techniques ; Potassium ; metabolism ; pharmacology ; Potassium Channels ; physiology ; Receptors, Cholinergic ; drug effects ; metabolism

Hearing loss is the most frequent sensorineural disorder worldwild, among which about 50% are caused by genetic factors. TMC1 is one of the common genes causing hereditary hearing loss. TMC1 mutations can cause pre-lingual profound/severe autosomal recessive (DFNB7/11) and post-lingual progressive autosomal dominant (DFNA36) non-syndromic hearing loss. Murine models studies show that TMC1, 2 are expressed in cochlea inner and outer hair cells and maintain normal mechanoelectrical transduction (MET) functions of the hair cells. A growing number of evidence indicate that TMC1, 2 are components of the MET complex. It is necessary to definite the precise distribution and exact function of TMC1, 2, because it is important to understand the regulating mechanism of auditory function.
Animals ; Cochlea ; metabolism ; Disease Models, Animal ; Hair Cells, Auditory, Outer ; metabolism ; Hearing Loss, Sensorineural ; genetics ; Humans ; Membrane Proteins ; genetics ; Mice ; Mutation

OBJECTIVETo investigate the relationship between caspase 12 activation and endoplasmic reticulum stress mediated apoptosis of guinea pig cochlea cells induced by intense noise.

METHODSThirty-two guinea pigs were randomly divided into 4 groups. The guinea pigs in the experiment groups were exposed to 4 kHz narrow band noise at 120 dB SPL for 4 h. After the noise expose for 1, 4, 14 days of the experiment guinea pigs, auditory brainstem response (ABR) of the guinea pigs on experiment and control groups were tested before decapitated. Four guinea pig's cochleae of every group were taken to paraffin section, and the rest was extracted the total protein. Apoptosis was tested by terminal deoxynucleotidyl transferase (TDT)-mediated deoxyuridine triphosphate (d-UTP) nick and labeling method (TUNEL) and transmission electron microscopy. Expression of caspase 12, Bip/GRP78 was tested by immunohistochemistry and Western blot methods.

RESULTSThe observation by transmission electron microscopy showed the features characteristic of apoptotic cells in the Corti and SGC of 1d after the noise expose, but no in the control. There were higher expressions of Tunel-Positive cells in the OHC, SGC and SV of experiment groups, and there was significant differences compared with the control group (P < 0.01). Protein levels of Bip/GRP78 and caspase 12 were risen up after noise exposed, and there all were significant differences compared with the control group (P < 0.01).

CONCLUSIONIntense noise causes cochlea cell lesion by inducing apoptosis to result in and caspase 12 induced endoplasmic reticulum stress-related apoptosis plays an important role in the procedure of apoptosis.

Animals ; Apoptosis ; Caspase 12 ; metabolism ; Cochlea ; cytology ; metabolism ; pathology ; Endoplasmic Reticulum ; metabolism ; Evoked Potentials, Auditory, Brain Stem ; Guinea Pigs ; Hair Cells, Auditory, Outer ; metabolism ; pathology ; Male ; Noise ; adverse effects

OBJECTIVE: Effects of ATP and acetylcholine (ACh) on intracellular Ca2+ concentrations ([Ca2+]i) and possible mechanism of Ca2+-induced Ca2+ release (CICR) of the isolated outer hair cells (OHCs) in the guinea pig cochlea were studied with confocal microscopy. METHOD: OHCs were isolated from guinea pig cochlea by enzymatic and mechanical methods. The effects of ATP, ACh, Ryanodine + ATP (or ACh) and Thapsigargin + ATP (or ACh) in the presence or absence of extracellular Ca2+ on [Ca2+]i in OHCs were examined by confocal microscopy. RESULT: In the presence of ATP, Ryanodine + ATP, Thapsigargin + ATP, ACh, Ryanodine + ACh and Thapsigargin + ACh increased [Ca2+]i and evoked an evident wave, respectively, the relative magnitude of fluorescence were 1.60 +/- 0.01(ATP), 1.644 +/- 0.005 (Ryanodine + ATP), 1.491 +/- 0.005 (Thapsigargin + ATP), 1.43 +/- 0.01 (ACh), 1.58 +/- 0.02 (Ryanodine + ACh), 1.398 +/- 0.003 (Thapsigargin + ACh) in OHCs in the presence of extracellular Ca2+ respectively. In the absence of extracellular Ca2+, ATP and Ryanodine + ATP induced a gradual and small [Ca2+]i wave, the relative magnitude of fluorescence were 1.341 +/- 0.006 and 1.386 +/- 0.008, however, ACh, Ryanodine + ACh, Thapsigargin + ACh and Thapsigargin + ATP can not induce wave but a gradual [Ca2+]i elevation. ACh can not increase [Ca2+]i. CONCLUSION In the presence of extracellular Ca2+, ATP and ACh increased [Ca2+]i in OHCs not only by Ca2+ influx through ion channel on cell membrane but also a release of Ca2+ from IP3-sensitive calcium reservoir and CICR. In the absence of extracellular Ca2+, ATP activated IP3 sensitive calcium reservoir and Ca2+ release through IP3 sensitive calcium reservoir, in turn CICR was induced. ACh can not activate IP3 sensitive calcium reservoir and CICR in the absence of extracellular Ca2+, therefore, the effect of ACh was dependent of extracellular Ca2+.
Acetylcholine ; pharmacology ; Adenosine Triphosphate ; pharmacology ; Animals ; Calcium ; metabolism ; Calcium Channels ; drug effects ; metabolism ; Cells, Cultured ; Cochlea ; cytology ; metabolism ; Guinea Pigs ; Hair Cells, Auditory, Outer ; metabolism ; Microscopy, Confocal ; Ryanodine ; pharmacology ; Thapsigargin ; pharmacology