Efferent synapses return to inner hair cells in the aging cochlea

Efferent innervation of the cochlea undergoes extensive modification early in development, but it is unclear if efferent synapses are modified by age, hearing loss, or both. Structural alterations in the cochlea affecting information transfer from the auditory periphery to the brain may contribute to age-related hearing deficits. We investigated changes to efferent innervation in the vicinity of inner hair cells (IHCs) in young and old C57BL/6 mice using transmission electron microscopy to reveal increased efferent innervation of IHCs in older animals. Efferent contacts on IHCs contained focal presynaptic accumulations of small vesicles. Synaptic vesicle size and shape were heterogeneous. Postsynaptic cisterns were occasionally observed. Increased IHC efferent innervation was associated with a smaller number of afferent synapses per IHC, increased outer hair cell loss, and elevated auditory brainstem response thresholds. Efferent axons also formed synapses on afferent dendrites but with a reduced prevalence in older animals. Age-related reduction of afferent activity may engage signaling pathways that support the return to an immature state of efferent innervation of the cochlea.     

ATP-induced current in isolated outer hair cells of guinea pig cochlea

1. Electrical and pharmacologic properties of ATP-induced current in outer hair cells isolated from guinea pig cochlea were investigated in the whole-cell recording mode by the use of a conventional patch-clamp technique. 2. Under current-clamp conditions, rapid application of ATP depolarized the outer hair cells resulting in an increase in conductance. The ATP-induced response did not show any desensitization during a continuous application. 3. At a holding potential of -70 mV, the ATP-induced inward current increased in a sigmoidal fashion over the concentration range between 3 microM and 1 mM. The half-maximum concentration (EC50) was 12 microM and the Hill coefficient was 0.93. 4. The ATP-induced current had a reversal potential near 6 mV, which was close to the theoretical value (1 mV) calculated from the Goldman-Hodgkin-Katz equation for permeable intra- and extracellular cations. 5. In the current-voltage (I-V) relationship for the ATP response, a slight inward-going rectification was observed at more positive potentials than the reversal potential. 6. The substitution of extracellular Na+ by equimolar choline+ shifted the reversal potential of the ATP-induced current to more negative values. The substitution of Cs+ in the internal solution by N-methyl-D-glucamine+ (NMG+) shifted it in the positive direction. The reversal potential of ATP-induced current was also shifted to positive values with increasing extracellular Ca2+ concentration. A decrease of intracellular Cl- by gluconate- did not affect the reversal potential, thereby indicating that the ATP-induced current is carried through a large cation channel.(ABSTRACT TRUNCATED AT 250 WORDS)     

庆大霉素中毒后豚鼠耳蜗毛细胞的再生

目的：通过庆大霉素中毒后不同时间豚鼠耳蜗毛细胞变化的观察，研究哺乳动物耳蜗毛细胞受损后能否再生。方法：将60只豚鼠随机分成庆大霉素组和生理盐水对照组。应用扫描电镜（SEM）技术并结合听脑干反应（ABR）测试，观察耳蜗毛细胞情况和ABR阀值变化。结果：庆大霉素组耳蜗毛细胞在30 d时ABR阈值有明显恢复，但未达到正常水平，同时耳蜗第三转有新生的静纤毛出现。结论：庆大霉素损伤后的毛细胞可以再生。     

Transient expression of an inwardly rectifying potassium conductance in developing inner and outer hair cells along the mouse cochlea

Inwardly rectifying K+ currents in inner and outer hair cells (IHCs, OHCs) were studied during post-natal development of the mouse cochlea. Hyperpolarizing steps from a holding potential of -64 mV induced a rapidly activating current in both cell types. This current showed strong inward rectification around the K+ equilibrium potential and, at potentials negative to -130 mV, partial inactivation. The activation range varied with extracellular K+ concentration. External application of Ba2+ and Cs+ reversibly blocked the elicited current. The results are consistent with the presence of an IK1-type inwardly rectifying potassium conductance in these cells. The maximum current was 60% larger in IHCs than in OHCs. In OHCs, but not IHCs, the amplitude of IK1 varied significantly with the cells' position along the cochlea. IK1 was maximal in cells located in the most basal region of the cochlea and its amplitude decreased in the apical coil. IK1 disappeared upon functional maturation: in OHCs at the end of the first postnatal week, and in IHCs at the onset of auditory function 12 days after birth. The current is active at the resting potential of the cells and plays a role in regulating the spiking behaviour characteristic of developing hair cells.     

Dll3 is expressed in developing hair cells in the mammalian cochlea.

Notch mediates the process of lateral inhibition that controls the production of hair cells in the inner ear. Hair cells are known to express Notch ligands Dll1 and Jag2, which signal through Notch1 in adjacent supporting cells. However, recent genetic and pharmacological studies indicate that the level of Notch-mediated lateral inhibition is greater than can be accounted for by Dll1 and Jag2. Here, we report that another Notch ligand, Dll3, is expressed in developing hair cells, in a pattern that overlaps that of Dll1 and Jag2. We analyzed the cochleae of Dll3(pu) mutant mice, but did not detect any abnormalities. However, earlier studies have demonstrated that there is functional redundancy among Notch ligands in cochlear development and loss of one ligand can be at least partially compensated for by another. Thus Dll3 may play a role in lateral inhibition similar to that of Dll1 and Jag2.     

Facilitating efferent inhibition of inner hair cells in the cochlea of the neonatal rat

Cholinergic brainstem neurones make inhibitory synapses on outer hair cells (OHCs) in the mature mammalian cochlea and on inner hair cells (IHCs) prior to the onset of hearing. We used electrical stimulation in an excised organ of Corti preparation to examine evoked release of acetylcholine (ACh) onto neonatal IHCs from these efferent fibres. Whole-cell voltage-clamp recording revealed that low frequency (0.25–1 Hz) electrical stimulation produced evoked inhibitory postsynaptic currents (IPSCs) at a relatively high fraction of failures (65%) and with mean amplitudes of about −20 pA at −90 mV, corresponding to a quantum content of ∼1. Evoked IPSCs had biphasic waveforms at −60 mV, were blocked reversibly by α-bungarotoxin and strychnine and are most likely mediated by the α9/α10 acetylcholine receptor, with subsequent activation of calcium-dependent potassium (SK2) channels. Paired pulse stimulation with intervals of 10–100 ms caused facilitation of 200–300% in the mean IPSC amplitude. A train of 10 pulses with an interpulse interval of 25 ms produced increasingly larger IPSCs with maximum amplitudes greater than −100 pA due to facilitation and summation throughout the train. Repetitive efferent stimulation at 5 Hz or higher hyperpolarized IHCs by 5–10 mV and could completely prevent the generation of calcium action potentials normally evoked by depolarizing current injection.     

Two classes of outer hair cells along the tonotopic axis of the cochlea

The molecular basis of high versus low frequency hearing loss and the differences in the sensitivity of outer hair cells depending on their cochlear localization are currently not understood. Here we demonstrate the existence of two different outer hair cell phenotypes along the cochlear axis. Outer hair cells in low frequency regions exhibit early sensitivity for loss of Ca(v)1.3 (alpha1 subunit 1.3 forming the class D L-type voltage-gated Ca(2+) channel), while high frequency regions display a progressive susceptibility for loss of the Ca(2+)-activated large conductance K(+) (BK) channel. Despite deafness, young Ca(v)1.3-deficient mice displayed distortion-product otoacoustic emissions (DPOAEs), indicating functional outer hair cells in the higher frequency range of the cochlea. Considering that DPOAEs are also found in the human deafness syndrome DFNB9 caused by mutations in the synaptic vesicle protein otoferlin, we tested the expression of otoferlin in outer hair cells. Surprisingly, otoferlin showed a distinct tonotopic expression pattern at both the mRNA and protein level. Otoferlin-expressing, Ca(v)1.3 deletion-sensitive outer hair cells in the low frequency range could be clearly separated from otoferlin-negative, BK deletion-sensitive outer hair cells in the high frequency range. In addition, BK deletion led to a higher noise vulnerability in low frequency regions, which are normally unaffected by the BK deletion alone, suggesting that BK currents are involved in survival mechanisms of outer hair cells under noise conditions. Our findings propose new mechanisms and candidate genes for explaining high and low frequency hearing loss.     

Calcium channel in isolated outer hair cells of guinea pig cochlea.

The physiological and pharmacological properties of the Ca2+ channel in outer hair cells (OHCs) freshly isolated from guinea pig cochlea were investigated using a whole-cell patch-clamp technique. The Ca2+ current (ICa) was activated from a membrane potential of -20 mV and reached peak value around +20 mV in external solution containing 20 mM Ca2+ at a holding potential of -70 mV. The peak amplitude of ICa increased in a hyperbolic manner with increasing extracellular Ca2+ concentration. The ion selectively was Ba2+ much greater than Ca2+ greater than or equal to Sr2+. It was concluded that the Ca2+ channel in OHCs of guinea pig is of the L-type.     

Calcium- and calmodulin-dependent inactivation of calcium channels in inner hair cells of the rat cochlea

Modulation of voltage-gated calcium channels was studied in inner hair cells (IHCs) in an ex vivo preparation of the apical turn of the rat organ of Corti. Whole cell voltage clamp in the presence of potassium channel blockers showed inward calcium currents with millisecond activation and deactivation kinetics. When temperature was raised from 22 to 37 degrees C, the calcium currents of immature IHCs [<12 days postnatal (P12)] increased threefold in amplitude, and developed more pronounced inactivation. This was determined to be calcium-dependent inactivation (CDI) on the basis of its reliance on external calcium (substitution with barium), sensitivity to internal calcium-buffering, and voltage dependence (reflecting the calcium driving force). After the onset of hearing at P12, IHC calcium current amplitude and the extent of inactivation were greatly reduced. Although smaller than in prehearing IHCs, CDI remained significant in the mature IHC near the resting membrane potential. CDI in mature IHCs was enhanced by application of the endoplasmic calcium pump blocker, benzo-hydroquinone. Conversely, CDI in immature IHCs was reduced by calmodulin inhibitors. Thus voltage-gated calcium channels in mammalian IHCs are subject to a calmodulin-mediated process of CDI. The extent of CDI depends on the balance of calcium buffering mechanisms and may be regulated by calmodulin-specific processes. CDI provides a means for the rate of spontaneous transmitter release to be adjusted to variations in hair cell resting potential and steady state calcium influx.     

雷公藤甲素对2型糖尿病小鼠耳蜗毛细胞的保护作用

目的：探讨雷公藤甲素对2 型糖尿病小鼠耳蜗毛细胞的保护作用及机制。方法：选取 Balb/c 雄性小鼠,随 机分为正常组、糖尿病组和雷公藤甲素组。制备2 型糖尿病小鼠模型后,雷公藤甲素组给予雷公藤甲素0.1 mg/kg 灌胃,正常组和糖尿病组小鼠均给予等体积生理盐水灌胃。观察3 组小鼠耳蜗毛细胞的变化,检测耳蜗毛细胞内 氧化应激相关信号通路、抗氧化蛋白及凋亡分子的表达变化。结果：糖尿病组和雷公藤甲素组耳蜗毛细胞均有不 同程度的丢失,且雷公藤甲素组耳蜗毛细胞的丢失率小于糖尿病组。雷公藤甲素组小鼠耳蜗毛细胞中的p-ERK、 E2 核因子相关因子2（Nrf2）、硫氧还蛋白（Trx）表达量及Erk 磷酸化水平高于糖尿病组, JNK磷酸化水平和凋 亡分子cleaved-caspase-3 的蛋白表达量低于糖尿病组。结论：雷公藤甲素可以减缓糖尿病小鼠耳蜗毛细胞凋亡, 其机制与上调ERK、Nrf2 和Trx 的表达,下调JNK信号,抑制凋亡分子cleaved-caspase-3 的表达有关,有望成为 治疗2 型糖尿病耳蜗病变的有效药物。     

A calcium-activated nonselective cationic channel in the basolateral membrane of outer hair cells of the guinea-pig cochlea

The patch-clamp technique was used to investigate ion channels in the basolateral perilymph-facing membrane of freshly isolated outer hair cells (OHCs) from the guinea-pig cochlea. These sensory cells probably determine, via their motile activity, the fine tuning of sound frequencies and the high sensitivity of the inner ear. A Ca²⁺-activated nonselective cationic channel was found in excised inside-out membrane patches. The current/voltage relationship was linear with a unit conductance of 26.3±0.3 pS (n=15) under symmetrical inger conditions. The channel excluded anions (P _Na/P _Cl=18 whereP _Na/P _Cl denotes the relative permeability of Na to Cl); it was equally permeant to the Na⁺ and K⁺ ions and exhibited a low permeability toN-methyl-D-glucamine and Ba²⁺ or Ca²⁺. Channel opening required a free Ca²⁺ concentration of about 10^–6 mol/l on the internal side of the membrane and the open probability (P _o) was maximal at 10^–3 mol/l (P _o=0.72±0.06,n=12). Adenosine 5mono-, tri- and di-phosphate reducedP _o to 29±14 (n=5), 42±10 (n=8) and 51±12 (n=5) % of controlP _o, respectively, when they were added at a concentration of 10^–3 mol/l to the internal side. The channel was partially blocked by flufenamic acid (10^–4 mol/l) and 3,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC, 10^–5 mol/l). This type of channel, together with Ca²⁺-activated K⁺ channels, might participate in the control of membrane potential and modulate the motility of OHCs.     

Effect of cyclopiazonic acid on membrane currents in isolated inner hair cells from guinea-pig cochlea

Cyclopiazonic acid (CPA) is a reticulum-like intracellular Ca(2+) store depletory, which raises intracellular Ca(2+) concentration. The effect of CPA on membrane currents in isolated inner hair cells (IHCs) from guinea-pig cochlea was investigated by the patch-clamp technique in the whole-cell configuration. Four out of eight IHCs showed an augmentation of the currents and the other four cells showed an inhibition of the currents by extracellular CPA application. The activation kinetics of outward currents were not changed by CPA. Three out of four IHCs obtained from the basal part of the cochlea demonstrated augmentation, whereas three out of four IHCs from the apical part demonstrated inhibition of the currents. This result suggests that Ca(2+)-activated currents were dominant in the basal IHCs of the cochlea.     

Differential expression of potassium currents in Deiters cells of the guinea pig cochlea

Among the supporting cells, Deiters cells are in intimate contact with outer hair cells (OHCs) in the inner ear. The aim of this study was to characterize the outward rectifying K⁺ current of Deiters cells in conjunction with cellular morphological characteristics. In the majority of cells, the K⁺ current had a biphasic inactivation kinetics (τ ₁ and τ ₂ were 2,735±90 (n=77) and 160±14 ms (n=72), respectively). The rapidly inactivating current component was more sensitive to Charybdotoxin (ChTx, 10 nM) block whereas the slowly inactivating current could be blocked more efficiently by tetraethylammonium (1 mM). All these point toward the existence of two distinct potassium channel types in these cells. Deiters cells attached to shorter OHCs had more voluminous, whereas those attached to longer OHCs had lanky cell bodies. The inactivation kinetics was slower in cells having corpulent cell bodies due to the increased proportion of the slowly inactivating current component (0.736±0.033, n=27) as compared to the one determined for lanky cells (0.522±0.023, n=36). The average peak K⁺ current was higher in Deiters cells connected to OHCs (5,417±541 pA, n=40) than in isolated ones (3,527±410, n=37). Deiters cells having different cell shapes and showing different K⁺ channel expression may contribute to the active mechanism of the cochlea to various degrees.Attila Szûcs and Sándor Somodi contributed equally to this work.     

链霉素耳中毒豚鼠耳蜗外毛细胞外向钾通道的变化

目的:探讨链霉素耳中毒豚鼠耳蜗外毛细胞外向钾通道的变化。方法:应用听觉脑干诱发电位和全细胞膜片钳制技术。结果:链霉素耳中毒豚鼠体重明显降低;在以2 kHz为主的click刺激时听阈明显升高;从链霉素耳中毒豚鼠中分离的外毛细胞数明显减少,且以直径较长细胞为主;链霉素显著降低豚鼠耳蜗外毛细胞延迟外向K⁺电流;链霉素耳中毒豚鼠耳蜗外毛细胞Ca²⁺敏感外向K⁺通道和延迟外向K⁺通道的反转电位与正常对照组相比没有差异。结论:链霉素对外毛细胞外向钾通道的阻断作用是其耳毒性的基础,但不是细胞致死的直接原因。     

Tmc1 is necessary for normal functional maturation and survival of inner and outer hair cells in the mouse cochlea

The deafness ( dn ) and Beethoven ( Bth ) mutant mice are models for profound congenital deafness (DFNB7/B11) and progressive hearing loss (DFNA36), respectively, caused by recessive and dominant mutations of transmembrane cochlear-expressed gene 1 ( TMC1 ), which encodes a transmembrane protein of unknown function. In the mouse cochlea Tmc1 is expressed in both outer (OHCs) and inner (IHCs) hair cells from early stages of development. Immature hair cells of mutant mice seem normal in appearance and biophysical properties. From around P8 for OHCs and P12 for IHCs, mutants fail to acquire ( dn / dn ) or show reduced expression ( Bth / Bth and, to a lesser extent Bth /+) of the K⁺ currents which contribute to their normal functional maturation (the BK-type current I _K,f in IHCs, and the delayed rectifier I _K,n in both cell types). Moreover, the exocytotic machinery in mutant IHCs does not develop normally as judged by the persistence of immature features of the Ca²⁺ current and exocytosis into adulthood. Mutant mice exhibited progressive hair cell damage and loss. The compound action potential (CAP) thresholds of Bth /+ mice were raised and correlated with the degree of hair cell loss. Homozygous mutants ( dn / dn and Bth / Bth ) never showed CAP responses, even at ages where many hair cells were still present in the apex of the cochlea, suggesting their hair cells never function normally. We propose that Tmc1 is involved in trafficking of molecules to the plasma membrane or serves as an intracellular regulatory signal for differentiation of immature hair cells into fully functional auditory receptors.