Expression of peripherin in human cochlea

The organ of Corti contains two different types of auditory receptors; the inner (IHCs) and outer (OHCs) hair cells. This dualism is further represented in their innervation, IHCs being innervated by type I neurons, and OHCs by type II neurons (in man, named small ganglion cells). Two efferent systems are also present. Here, we have analyzed the expression of the 57-kDa neuron-specific intermediate filament protein peripherin (PP) in human cochlea. In the human organ of Corti, PP seems to be specifically expressed in OHC afferents. Small or type II spiral ganglion cell bodies also intensely express PP. Thus, PP can be used as a marker for the characterization of the innervation of the OHC system in man.     

P物质、脑啡肽在豚鼠耳蜗的分布——免疫组织化学观察

本研究应用免疫组织化学方法系统地观察了P物质(SP)、亮氨酸脑啡肽(L-ENK)在豚鼠耳蜗的分布以及SP、L-ENK免疫反应阳性神经纤维与Corti's器毛细胞之间的关系,结果表明:SP的免疫反应活性(SP-IR)存在于耳蜗螺旋神经节的部分神经细胞及传入神经纤维中,在Corti's器的毛细胞下方亦可见SP免疫反应阳性纤维;L-ENK的免疫反应活性(ENK-IR)存在于耳蜗的传出神经纤维中。节内螺旋束、内螺旋束、隧道螺旋束、横贯纤维均含有大量的L-ENK免疫反应阳性纤维,Cort's器中的L-ENK免疫反应阳性终末与毛细胞之间具有密切接触,由此提示,SP可能为听觉初级传入神经递质之一;L-ENK作为传出神经递质或调质对听觉传入起调控作用。     

Power Efficiency of Outer Hair Cell Somatic Electromotility

Cochlear outer hair cells (OHCs) are fast biological motors that serve to enhance the vibration of the organ of Corti and increase the sensitivity of the inner ear to sound. Exactly how OHCs produce useful mechanical power at auditory frequencies, given their intrinsic biophysical properties, has been a subject of considerable debate. To address this we formulated a mathematical model of the OHC based on first principles and analyzed the power conversion efficiency in the frequency domain. The model includes a mixture-composite constitutive model of the active lateral wall and spatially distributed electro-mechanical fields. The analysis predicts that: 1) the peak power efficiency is likely to be tuned to a specific frequency, dependent upon OHC length, and this tuning may contribute to the place principle and frequency selectivity in the cochlea; 2) the OHC power output can be detuned and attenuated by increasing the basal conductance of the cell, a parameter likely controlled by the brain via the efferent system; and 3) power output efficiency is limited by mechanical properties of the load, thus suggesting that impedance of the organ of Corti may be matched regionally to the OHC. The high power efficiency, tuning, and efferent control of outer hair cells are the direct result of biophysical properties of the cells, thus providing the physical basis for the remarkable sensitivity and selectivity of hearing.     

Expression of alpha and beta parvalbumin is differentially regulated in the rat organ of corti during development

The expression of two calcium-binding proteins of the parvalbumin (PV) family, the alpha isoform (alphaPV) and the beta isoform known as oncomodulin (OM), was investigated in the rat cochlea during postnatal development and related to cholinergic efferent innervation. Using RT-PCR analysis, we found that OM expression begins between postnatal day 2 (P2) and P4, and peaks as early as P10, while alphaPV mRNA begins expression before birth and remains highly expressed into the adult period. Both in situ hybridization and immunoreactivity confirm that OM is uniquely expressed by the outer hair cells (OHCs) in the rat cochlea and occurs after efferent innervation along the cochlear spiral between P2 and P4. In contrast to OM expression, alphaPV immunoreactivity is expressed in both inner hair cells (IHCs) and OHCs at birth. Following olivocochlear efferent innervation, OHCs demonstrate weak OM immunoreactivity beginning at P5 and diminished alphaPV immunoreactivity after P10. In organ cultures isolated prior to the efferent innervation of OHCs, OM immunoreactivity failed to develop in OHCs, but alphaPV immunoreactivity remained present in both IHCs and OHCs. In contrast, organ cultures isolated after efferent innervation of OHCs show OHCs with low levels of OM immunoreactivity and high levels of alphaPV immunoreactivity. This study suggests that OM and alphaPV are differentially regulated in OHCs during cochlear development. Our findings further raise the possibility that the expression of PV proteins in OHCs may be influenced by efferent innervation.     

Expression of α and β parvalbumin is differentially regulated in the rat organ of corti during development

The expression of two calcium‐binding proteins of the parvalbumin (PV) family, the α isoform (αPV) and the β isoform known as oncomodulin (OM), was investigated in the rat cochlea during postnatal development and related to cholinergic efferent innervation. Using RT‐PCR analysis, we found that OM expression begins between postnatal day 2 (P2) and P4, and peaks as early as P10, while αPV mRNA begins expression before birth and remains highly expressed into the adult period. Both in situ hybridization and immunoreactivity confirm that OM is uniquely expressed by the outer hair cells (OHCs) in the rat cochlea and occurs after efferent innervation along the cochlear spiral between P2 and P4. In contrast to OM expression, αPV immunoreactivity is expressed in both inner hair cells (IHCs) and OHCs at birth. Following olivocochlear efferent innervation, OHCs demonstrate weak OM immunoreactivity beginning at P5 and diminished αPV immunoreactivity after P10. In organ cultures isolated prior to the efferent innervation of OHCs, OM immunoreactivity failed to develop in OHCs, but αPV immunoreactivity remained present in both IHCs and OHCs. In contrast, organ cultures isolated after efferent innervation of OHCs show OHCs with low levels of OM immunoreactivity and high levels of αPV immunoreactivity. This study suggests that OM and αPV are differentially regulated in OHCs during cochlear development. Our findings further raise the possibility that the expression of PV proteins in OHCs may be influenced by efferent innervation. © 2003 Wiley Periodicals, Inc. J Neurobiol 58: 479–492, 2004     

Morphological and molecular changes in the inner hair cell region of the rat cochlea after amikacin treatment

This study investigates the morphological and molecular changes that occur in the inner hair cell area of the rat cochlea following aminoglycoside treatment. Rats were injected daily with 500 mg/kg of amikacin between postnatal day 9 (PND9) and PND16. Cochleae were examined at PND16 to PND120 using both scanning and transmission electron microscopy and molecular fluorescent labeling. The inner hair cells showed obvious signs of apoptosis in response to amikacin treatment and most of them were missing by one week after the end of the aminoglycoside exposure period. Concomitantly, the epithelium became scarred as the surrounding supporting cells expanded and filled the space vacated by the missing IHCs. The mid-basolateral region of these modified supporting cells was surrounded by many afferent and efferent terminals. However, these cells expressed neither calbindin nor SNAP25, proteins that are both expressed by IHCs in the normal, untreated organ of Corti in the rat. In addition, these supporting cells remained attached to the basal lamina by a thin cytoplasmic process. The supporting cells surrounding the inner hair cells therefore appear unable to convert directly into inner hair cells following aminoglycoside induced hair-cell loss but may be able to provide trophic support for the remaining afferent and efferent neurites.     

Cholinergic efferent synaptic transmission regulates the maturation of auditory hair cell ribbon synapses

Spontaneous electrical activity generated by developing sensory cells and neurons is crucial for the maturation of neural circuits. The full maturation of mammalian auditory inner hair cells (IHCs) depends on patterns of spontaneous action potentials during a ‘critical period’ of development. The intrinsic spiking activity of IHCs can be modulated by inhibitory input from cholinergic efferent fibres descending from the brainstem, which transiently innervate immature IHCs. However, it remains unknown whether this transient efferent input to developing IHCs is required for their functional maturation. We used a mouse model that lacks the α9-nicotinic acetylcholine receptor subunit (α9nAChR) in IHCs and another lacking synaptotagmin-2 in the efferent terminals to remove or reduce efferent input to IHCs, respectively. We found that the efferent system is required for the developmental linearization of the Ca²⁺-sensitivity of vesicle fusion at IHC ribbon synapses, without affecting their general cell development. This provides the first direct evidence that the efferent system, by modulating IHC electrical activity, is required for the maturation of the IHC synaptic machinery. The central control of sensory cell development is unique among sensory systems.     

Ba2+ currents in inner and outer hair cells of mice lacking the voltage-dependent Ca2+ channel subunits β3 or β4

Voltage-activated Ca2+ channels comprise complexes of a pore-forming Cavα1 and auxiliary subunits Cavβ, Cavα2δ and sometimes Cavγ. The intracellular Cavβ subunit assists in trafficking and surface expression of the Cavα1 subunit and can modulate biophysical properties of the Ca2+ channel. Four genes, Cavβ1-4, exist which confer different properties to Ca2+ currents through the various Cavα1 subunits. Ca2+ currents in cochlear inner (IHC) and outer hair cells (OHC) serving synaptic transmission flow predominantly through the L type Cavα1 subunit Cav1.3, but associated Cavβ subunits are unknown. In the organ of Corti, we found mRNA and protein for all four Cavβ subunits including Cavβ2, but clear assignment of the Cavβ1 4 immunolabelling with hair cells or nerve fibers was difficult. We analyzed Cavβ3 knockout (Cavβ3 / ) and Cavβ4 mutant mice (Cavβ4lh/lh), which had normal hearing. Recording voltage-activated Ba2+ currents from hair cells of the two mouse models revealed distinct significant changes of cell size and Ba2+ current properties compared with their wildtype controls. Neonatal Cavβ4lh/lh IHCs showed reduced membrane capacitances and changes in the voltage dependence and kinetics of current activation, whereas mature IHCs had reduced peak currents compared with Cavβ4wt, altogether indicating the presence of Cavβ4 in IHCs. Ba2+ currents of Cavβ3 / OHCs showed largely reduced amplitudes, changes in the voltage dependence and kinetics of Ba2+ current activation, and increased inactivation compared with Cavβ3wt, pointing to a role of Cavβ3 for OHCs. These results indicate that neither Cavβ3 nor Cavβ4 are indispensable for hair cell Ca2+ currents but contribute to the overall current properties.     

Imaging electrically evoked micromechanical motion within the organ of corti of the excised gerbil cochlea

The outer hair cell (OHC) of the mammalian inner ear exhibits an unusual form of somatic motility that can follow membrane-potential changes at acoustic frequencies. The cellular forces that produce this motility are believed to amplify the motion of the cochlear partition, thereby playing a key role in increasing hearing sensitivity. To better understand the role of OHC somatic motility in cochlear micromechanics, we developed an excised cochlea preparation to visualize simultaneously the electrically-evoked motion of hundreds of cells within the organ of Corti (OC). The motion was captured using stroboscopic video microscopy and quantified using cross-correlation techniques. The OC motion at approximately 2-6 octaves below the characteristic frequency of the region was complex: OHC, Deiter's cell, and Hensen's cell motion were hundreds of times larger than the tectorial membrane, reticular lamina (RL), and pillar cell motion; the inner rows of OHCs moved antiphasic to the outer row; OHCs pivoted about the RL; and Hensen's cells followed the motion of the outer row of OHCs. Our results suggest that the effective stimulus to the inner hair cell hair bundles results not from a simple OC lever action, as assumed by classical models, but by a complex internal motion coupled to the RL.     

Immunolocalization of parvalbumin in two glutamatergic cell types of the guinea pig cochlea: inner hair cells and spinal ganglion neurons

Immunoreactions to a monoclonal antibody raised against parvalbumin, a calcium-binding protein, have been detected in the inner hair cells of the organ of Corti and in the spiral ganglion neurons connected to them (type I neurons). Both cell types probably use an excitatory amino acid as a neurotransmitter (glutamate and/or aspartate). No immunoreactivity was found within the second sensory cell type (outer hair cells) nor in the olivocochlear (efferent) fibers or endings in the cochlea. In the central nervous system, parvalbumin may be involved in calcium-dependent mechanisms leading to neurotransmitter release. It could thus be hypothesized that parvalbumin also have similar implications at the level of the inner hair cell and type I neuron synapses. Additional functions could also be hypothesized for this protein in the cochlea. Within the inner hair cells, parvalbumin may be involved in the ionic regulation following potassium entry during the transduction process. Within type I neurons, by buffering sudden increases in the intracellular calcium concentration, it may allow an adaptation of the firing rate to variations in the intensity of sound stimuli.     

Optimal Electrical Properties of Outer Hair Cells Ensure Cochlear Amplification

The organ of Corti (OC) is the auditory epithelium of the mammalian cochlea comprising sensory hair cells and supporting cells riding on the basilar membrane. The outer hair cells (OHCs) are cellular actuators that amplify small sound-induced vibrations for transmission to the inner hair cells. We developed a finite element model of the OC that incorporates the complex OC geometry and force generation by OHCs originating from active hair bundle motion due to gating of the transducer channels and somatic contractility due to the membrane protein prestin. The model also incorporates realistic OHC electrical properties. It explains the complex vibration modes of the OC and reproduces recent measurements of the phase difference between the top and the bottom surface vibrations of the OC. Simulations of an individual OHC show that the OHC somatic motility lags the hair bundle displacement by ∼90 degrees. Prestin-driven contractions of the OHCs cause the top and bottom surfaces of the OC to move in opposite directions. Combined with the OC mechanics, this results in ∼90 degrees phase difference between the OC top and bottom surface vibration. An appropriate electrical time constant for the OHC membrane is necessary to achieve the phase relationship between OC vibrations and OHC actuations. When the OHC electrical frequency characteristics are too high or too low, the OHCs do not exert force with the correct phase to the OC mechanics so that they cannot amplify. We conclude that the components of OHC forward and reverse transduction are crucial for setting the phase relations needed for amplification.     

用共聚焦显微镜观察ACh及ATP对豚鼠耳蜗外毛细胞Ca^2＋的调控

用激光扫描共聚焦显微镜研究了一般公认的耳蜗传出神经递质乙酰胆碱（ＡＣｈ）和三磷酸腺苷（ＡＴＰ）对豚鼠耳蜗外毛细胞（ＯＨＣｓ）胞内游离Ｃａ＾２＋浓度（Ｃａ＾２＋）的作用,ＯＨＣｓ用Ｃａ＾２＋敏感荧光染料Ｆｌｕｏ－３着色,胞内Ｃａ＾２＋的分布以细胞底部稍强。ＡＣｈ在ＯＨＣ底部引起Ｃａ＾２＋的缓慢上长并维持在一个较高水平。ＡＴＰ在整个ＯＨＣ引起一个急剧的Ｃａ＾２＋升高,升高幅度在ＯＨＣ顶部最大。随着ＡＴ     

An ultrastructural study of the development of afferent and efferent synapses on outer hair cells of the guinea pig organ of Corti

The guinea pig organ of Corti was studied using transmission electron microscopy, the second turn of the cochlea being examined at various ages between 20 days before birth and 30 days postnatal. Outer hair cells were examined at each of these ages. At all ages studied, the efferent (presynaptic) terminals are large and are packed with synaptic vesicles, whereas the afferent (postsynaptic) terminals are generally smaller, with a relatively small number of vesicles. During development, the subsynaptic cistern changes from a fragmented, diffuse profile extending over 50-70% of the length of the efferent contact zones, to a continuous, compact structure spanning neighbouring synapses. Synaptic vesicles in the efferent terminals are predominantly rounded in early development, flattened vesicles appearing postnatally. The synaptic bodies at afferent synapses do not change noticeably during development. Quantitative analysis revealed that the area of efferent terminals and the length of their active zone increase with increasing age, the same parameters decreasing in afferent terminals. Synaptic vesicles in the efferent terminals decrease in diameter, but remain constant in afferent terminals, with increasing age. The number of hair cell membrane invaginations decreases as development proceeds.     

Fgf8 induces pillar cell fate and regulates cellular patterning in the mammalian cochlea

The mammalian auditory sensory epithelium (the organ of Corti) contains a number of unique cell types that are arranged in ordered rows. Two of these cell types, inner and outer pillar cells (PCs), are arranged in adjacent rows that form a boundary between a single row of inner hair cells and three rows of outer hair cells (OHCs). PCs are required for auditory function, as mice lacking PCs owing to a mutation in Fgfr3 are deaf. Here, using in vitro and in vivo techniques, we demonstrate that an Fgf8 signal arising from the inner hair cells is the key component in an inductive pathway that regulates the number, position and rate of development of PCs. Deletion of Fgf8 or inhibition of binding between Fgf8 and Fgfr3 leads to defects in PC development, whereas overexpression of Fgf8 or exogenous Fgfr3 activation induces ectopic PC formation and inhibits OHC development. These results suggest that Fgf8-Fgfr3 interactions regulate cellular patterning within the organ of Corti through the induction of one cell fate (PC) and simultaneous inhibition of an alternate fate (OHC) in separate progenitor cells. Some of the effects of both inhibition and overactivation of the Fgf8-Fgfr3 signaling pathway are reversible, suggesting that PC differentiation is dependent upon constant activation of Fgfr3 by Fgf8. These results suggest that PCs might exist in a transient state of differentiation that makes them potential targets for regenerative therapies.     

Electron microscopic examination of the orexin immunoreactivity in the dorsal raphe nucleus

The ultrastructure and the synaptic relationships of the orexin-A-like immunoreactive fibers in the dorsal raphe nucleus were examined with an immunoelectron microscopic method. At the electron microscopic level, most of the immunoreactive fibers, a varicosity appearance at the light microscopic level, were found as axon terminals. The large dense-cored vesicles contained in the immunoreactive axon terminals were the most intensely immunostained organellae. These axon terminals were often found to make synapses. While the axo-dendritic synapses were usually asymmetric in appearance, the axo-somatic synapses were symmetric. Orexin-A-like immunoreactive processes with no synaptic vesicles were also found. These processes often received asymmetric synapses. With less frequency, the synapses were found between the orexin-like immunoreactive processes. The results suggest that the orexin peptides are stored in the large dense-cored vesicles; the orexin-containing fibers may have influences on the physiological activities of the dorsal raphe nucleus through direct synaptic relationships.     

Effects of SERCA and PMCA inhibitors on the survival of rat cochlear hair cells during ischemia in vitro

An important mechanism underlying cochlear hair cell (HC) susceptibility to hypoxia/ischemia is the influx of Ca(2+). Two main ATP-dependent mechanisms contribute to maintaining low Ca(2+) levels: uptake of Ca(2+) into intracellular stores via smooth endoplasmic reticulum calcium ATPase (SERCA) and extrusion of Ca(2+) via plasma membrane calcium ATPase (PMCA). The effects of the SERCA inhibitors thapsigargin (10 nM-10 microM) and cyclopiazonic acid (CPA; 10-50 microM) and of the PMCA blockers eosin (1.5-10 microM) and o-vanadate (1-5 mM) on inner and outer hair cells (IHCs/OHCs) were examined in normoxia and ischemia using an in vitro model of the newborn rat cochlea. Exposure of the cultures to ischemia resulted in a significant loss of HCs. Thapsigargin and CPA had no effect. Eosin decreased the numbers of IHCs and OHCs by up to 25 % in normoxia and significantly aggravated the ischemia-induced damage to IHCs at 5 and 10 microM and to OHCs at 10 microM. o-Vanadate had no effect on IHC and OHC counts in normoxia, but aggravated the ischemia-induced HC loss in a dose-dependent manner. The effects of eosin and o-vanadate indicate that PMCA has an important role to play in protecting the HCs from ischemic cell death.     

Electromotility of outer hair cells from the cochlea of the echolocating bat,Carollia perspicillata

Isolated outer hair cells (OHCs) and explants ot the organ of Corti were obtained from the cochlea of the echolocating bat, Carollia perspicillata, whose hearing range extends up to about 100 kHz. The OHCs were about 10–30 m long and produced resting potentials between-30 to -69 mV. During stimulation with a sinusoidal extracellular voltage field (voltage gradient of 2 mV/m) cyclic length changes were observed in isolated OHCs. The displacements were most prominent at the level of the cell nucleus and the cuticular plate. In the organ of Corti explants, the extracellular electric field induced a radial movement of the cuticular plate which was observed using video subtraction and photodiode techniques. Maximum displacements of about 0.3–0.8 m were elicited by stimulus frequencies below 100 Hz. The displacement amplitude decreased towards the noise level of about 10–30 nm for stimulus frequencies between 100–500 Hz, both in apical and basal explants. This compares well with data from the guinea pig, where OHC motility induced by extracellular electrical stimulation exhibits a low pass characteristic with a corner frequency below 1 kHz. The data indicate that fast OHC movements presumably are quite small at ultrasonic frequencies and it remains to be solved how they participate in amplifying and sharpening cochlear responses in vivo.Abbreviations BM basilar membrane - FFT fast Fourier Transfer - IHC inner hair cell - OHC outer hair cell     

Deformation of the Outer Hair Cells and the Accumulation of Caveolin-2 in Connexin 26 Deficient Mice

BackgroundMutations in GJB2, which encodes connexin 26 (Cx26), a cochlear gap junction protein, represent a major cause of pre-lingual, non-syndromic deafness. The degeneration of the organ of Corti observed in Cx26 mutant—associated deafness is thought to be a secondary pathology of hearing loss. Here we focused on abnormal development of the organ of Corti followed by degeneration including outer hair cell (OHC) loss.MethodsWe investigated the crucial factors involved in late-onset degeneration and loss of OHC by ultrastructural observation, immunohistochemistry and protein analysis in our Cx26-deficient mice (Cx26^f/fP0Cre).ResultsIn ultrastructural observations of Cx26^f/fP0Cre mice, OHCs changed shape irregularly, and several folds or notches were observed in the plasma membrane. Furthermore, the mutant OHCs had a flat surface compared with the characteristic wavy surface structure of OHCs of normal mice. Protein analysis revealed an increased protein level of caveolin-2 (CAV2) in Cx26^f/fP0Cre mouse cochlea. In immunohistochemistry, a remarkable accumulation of CAV2 was observed in Cx26^f/fP0Cre mice. In particular, this accumulation of CAV2 was mainly observed around OHCs, and furthermore this accumulation was observed around the shrunken site of OHCs with an abnormal hourglass-like shape.ConclusionsThe deformation of OHCs and the accumulation of CAV2 in the organ of Corti may play a crucial role in the progression of, or secondary OHC loss in, GJB2-associated deafness. Investigation of these molecular pathways, including those involving CAV2, may contribute to the elucidation of a new pathogenic mechanism of GJB2-associated deafness and identify effective targets for new therapies.     

Regulation of outer hair cell cytoskeletal stiffness by intracellular Ca2+: underlying mechanism and implications for cochlear mechanics

Two Ca(2+)-dependent mechanisms have been proposed to regulate the mechanical properties of outer hair cells (OHCs), the sensory-motor receptors of the mammalian cochlea. One involves the efferent neurotransmitter, acetylcholine, decreasing OHC axial stiffness. The other depends on elevation of intracellular free Ca(2+) concentration ([Ca(2+)](i)) resulting in OHC elongation, a process known as Ca(2+)-dependent slow motility. Here we provide evidence that both these phenomena share a common mechanism. In whole-cell patch-clamp conditions, a fast increase of [Ca(2+)](i) by UV-photolysis of caged Ca(2+) or by extracellular application of Ca(2+)-ionophore, ionomycin, produced relatively slow (time constant approximately 20s) cell elongation. When OHCs were partially collapsed by applying minimal negative pressure through the patch pipette, elevation of the [Ca(2+)](i) up to millimole levels (estimated by Fura-2) was unable to restore the cylindrical shape of the OHC. Stiffness measurements with vibrating elastic probes showed that the increase of [Ca(2+)](i) causes a decrease of OHC axial stiffness, with time course similar to that of the Ca(2+)-dependent elongation, without developing any measurable force. We concluded that, contrary to a previous proposal, Ca(2+)-induced OHC elongation is unlikely to be driven by circumferential contraction of the lateral wall, but is more likely a passive mechanical reaction of the turgid OHC to Ca(2+)-induced decrease of axial stiffness. This may be the key phenomenon for controlling gain and operating point of the cochlear amplifier.