豚鼠耳蜗单离Hensen细胞的分离技术及其钾通道

目的　探讨适合膜片钳技术的单离Hensen细胞的分离及对其钾通道的研究。方法　取豚鼠耳蜗 ,获得Corti器 ,采用酶消化和机械分离方法对Hensen细胞进行分离 ,并采用传统全细胞膜片钳技术 ,记录单离Hensen细胞的钾电流。结果　每只耳蜗可以获得活性良好的单离Hensen细胞 12± 3个。Hensen细胞的钾电流呈明显的外向整流性 ,只有迟电流 (IK) ,没有峰电流 (IA)。结论　酶消化结合机械分离的方法可获得适用于膜片钳技术研究的单离Hensen细胞 ,并用于讨论了所记录到的Hensen细胞钾电流     

豚鼠耳蜗单离外毛细胞游离钙的胆碱能调控

目的　观察胆碱能兴奋剂和拮抗剂对豚鼠耳蜗单离外毛细胞 (OHC)内游离钙浓度 ([Ca2 + ]i)的影响 ,探讨OHC内 [Ca2 + ]i的胆碱能调控机制。方法　健康豚鼠 15只处死 ,Hanks液中分离活性单离OHC ,Fluo - 3/AM负载 30min。分 7组进行不同干预 ,包括乙酰胆碱 (ACH ,胆碱能M和N受体兴奋剂 )、毒蕈碱 (M受体兴奋剂 )、阿托品 (M受体拮抗剂 )、庆大霉素 (N受体拮抗剂 )、加Hanks液对照组和不加干预对照组 ;另 1组为dHanks液中加ACh。用激光扫描共聚焦显微镜 (Bio-Rad ,英国 )观察OHC内 [Ca2 + ]i的变化。结果　Hanks液中不加干预试剂、加毒蕈碱、加Hanks液对照和无钙dHanks液中加ACh等 4组 ,[Ca2 + ]i在 2 5～ 30min内升幅 <1.10倍。Hanks液中加ACh组 [Ca2 + ]i最高升幅平均为 3.0 9倍 ;阿托品 +ACh组 [Ca2 + ]i为 2 .92倍 ;庆大霉素 +ACh组[Ca2 + ]i为 1.81倍。结论　豚鼠耳蜗传出神经递质ACh通过兴奋N受体 ,开放OHC的细胞膜钙离子通道 ,使细胞外Ca2 + 内流外毛细胞内 ;[Ca2 + ]i增加需要细胞外液中钙离子存在。     

乙酰胆碱对豚鼠外毛细胞的电压依赖性外向整流钾电流的影响

目的 观察乙酰胆碱（ＡＣｈ）对不同长度豚鼠耳蜗外毛细胞（ＯＨＣ）电压依赖性外向整流钾电流的影响，分析 ＡＣｈ对钾电流激活动力学的影响。方法 全细胞膜片钳技术。结果１００μｍｏｌ／Ｌ的ＡＣｈ对短ＯＨＣ电压依赖性外向整流钾电流的影响较大，刺激电压为５０ｍＶ时，最大外向电流的幅度增加了３４．８％。ＡＣｈ对峰电流的影响大于稳态电流，改变了外向整流钾电流的动力学特征。ＡＣｈ将ＯＨＣ的零电流电位向超极化方向移位约５ｍＶ。1００μｍｏｌ／Ｌ的ＡＣｈ使ＯＨＣ电压依赖性外向整流钾电流的激活动力学发生改变，Ｖ（１／２）＝（－５２．３８±３．９８）ｍＶ，较作用前明显超极化，激活的电压敏感性也提高，Ｓ＝（４０±４．１４）ｍＶ（ｎ＝５）。结论ＡＣｈ增加了ＯＨＣ电压依赖性外向整流钾通道的电导，使通道的激活电压向超极化方向移位。ＡＣｈ的作用是使ＯＨＣ超极化。     

Potassium current properties in apical and basal inner hair cells from guinea-pig cochlea

Inner hair cells (IHCs) of guinea-pigs were separately isolated from the apical and basal turn and the potassium currents were measured by the whole-cell voltage-clamp technique. The potassium current flows through two types of membrane conductance: a fast (I_k,f), tetraethylammonium (TEA)-sensitive conductance and a slow (I_k,s), TEA-resistant conductance. Membrane conductance demonstrated no significant differences between apical IHCs and basal IHCs. Reversal potentials were −65±2 mV and −68±5 mV in apical and basal IHCs, respectively. The rate of outward current activation was voltage dependent and faster in basal IHCs than in apical IHCs. TEA effect was stronger on basal IHCs than on apical IHCs, suggesting that I_k,f is dominant in basal IHCs.     

噪声暴露对豚鼠耳蜗内毛细胞谷氨酸样免疫反应的影响

目的：了解噪声暴露对豚鼠耳蜗内毛细胞谷氨酸样免疫反应的影响，探讨噪声暴露引起耳蜗传入神经损伤的谷氨酸兴奋毒机制。方法：采用免疫组织化学方法结合计算机图像分析，观察噪声暴露后不同时间豚鼠耳蜗内毛细胞谷氨酸样免疫反应的改变。结果：在噪声后8小时，豚鼠耳蜗内毛细胞谷氨酸样免疫反应减弱。结论：噪声可引起耳蜗内毛细胞传入神经递质谷氨酸的过度释放，继而对耳蜗传入神经产生兴奋毒损伤，这可能是噪声后耳蜗传入神经损伤的机制之一。     

豚鼠耳蜗单离外毛细胞钙波的观察

目的：观察豚鼠耳蜗单离外毛细胞（outer hair cell,OHC)内游离钙浓度（[Ca^2]i)是否存在波动，即有无钙波。方法：健康豚鼠10只断头处死，在无Ca^2的dHanks液和含Ca^2的Hanks液中酶－机械法分离获得单离活性OHC，钙荧光抗体Fluo-3孵育后，分别加入乙酰胆碱或空白对照，用MRC－1024型激光扫描共聚焦显微镜（Bio-Rad，英国）观察OHC约50min内[Ca^2]i的变化。结果：dHanks液中加ACh组的5个OHC，[Ca^2]i呈明显的规律性的波动，即产生了钙波，钙波的周期约600s;Hanks液中加ACh组的6个OHC的[Ca^2]i均迅速升高，无钙波出现，在dHanks液和Hanks液中不加ACh分别观察了3个OHC，[Ca^2]i呈平坦型，无钙波，结论：ACh能刺激诱发豚鼠耳蜗单离OHC产生钙波。     

豚鼠耳蜗单离外毛细胞和Deiters细胞的钾离子电流及其生理功能

目的 研究豚鼠耳蜗单离外毛细胞(OHC)和Deiters细胞的钾离子电流及其特性，观察乙酰胆碱(ACh)和三磷酸腺苷(ATP)对离子电流的影响。方法 运用全细胞膜片钳技术，分别记录单离OHC和Deiters细胞的离子电流。结果 OHC的长度不同，其钾离子电流表达具有差异性，短OHC具有较大外向电流和内向电流。ACh对短OHC钾离子电流的影响较大，其作用是使0HC超极化。ATP介导的0HC内向电流是非选择性的阳离子电流。ATP介导Deiters细胞出现内向离子电流。结论 K^ 电流在OHC的电谐振中非常重要。Deiters细胞能够通过减少K^ 外流、增加K^ 内流来缓冲周围环境中K^ 浓度的变化。ACh和ATP对OHC离子电流的影响在耳蜗机制中起重要作用。     

A Biophysical Model of the Inner Hair Cell: The Contribution of Potassium Currents to Peripheral Auditory Compression

The term peripheral auditory compression refers to the fact that the whole range of audible sound pressure levels is mapped into a narrower range of auditory nerve responses. Peripheral compression is the by-product of independent compressive processes occurring at the level of the basilar membrane, the inner hair cell (IHC), and the auditory nerve synapse. Here, an electrical-circuit equivalent of an IHC is used to look into the compression contributed by the IHC. The model includes a mechanically driven transducer potassium (K⁺) conductance and two time- and voltage-dependent basolateral K⁺ conductances: one with fast and one with slow kinetics. Special attention is paid to faithfully implement the activation kinetics of these basolateral conductances. Optimum model parameters are provided to account for previously reported in vitro observations that demonstrate the compression associated with the gating of the transducer and of the basolateral channels. Without having to readjust its parameters, the model also accounts for the in vivo nonlinear IHC transfer characteristics. Model simulations are then used to investigate the relative contribution of the transducer and basolateral K⁺ currents to the nonlinear IHC input/output functions in vivo. The simulations suggest that the voltage-dependent activation of the basolateral currents compresses the DC potential for stereocilia displacements above approximately 5 nm. The degree of compression exceeds 2-to-1 and is similar for all stimulation frequencies. The AC potential is compressed in a similar way, but only for frequencies below 800 Hz. The simulations further suggest that the nonlinear gating of the transducer current is responsible for the expansive growth of the DC potential with increasing sound level (slope of 2 dB/dB) at low sound pressure levels.Both authors contributed equally to this work.     

回声定位蝙蝠耳蜗听黄斑区毛细胞形态研究

目的观察蝙蝠耳蜗听黄斑毛细胞的特化形态结构,以探讨听黄斑区毛细胞对蝙蝠回声定位回声频率的敏锐调谐、放大和频率选择的作用。方法选用听力正常的蹄蝠(22只)、菊头蝠(64只)和犬吻蝠(10只),分别检测其ABR各频率反应阈,对耳蜗基底膜毛细胞进行扫描电镜和透射电镜观察。结果三种蝙蝠ABR反应阈值除最佳频率外,均在35 dB SPL以上,菊头蝠、蹄蝠、犬吻蝠反应幅值最大及最敏锐调谐频率分别为83～86、60～62、20～28 kHz。回声定位蝙蝠耳蜗听黄斑区外毛细胞胞体(OHC)呈纺锤形或烧瓶形,明显不同于非回声定位哺乳动物的长圆柱形外毛细胞形态。结论回声定位蝙蝠耳蜗听黄斑OHC的形态特化特征支持其OHC作为耳蜗阻尼机制的结论,OHC的这种形态特化与其他耳蜗微机械结构的匹配有利于耳蜗前行波的传导。     

外源性谷氨酸对豚鼠耳蜗内、外毛细胞易损性的比较

目的观察外源性谷氨酸对豚鼠耳蜗电位及耳蜗内、外毛细胞的影响。方法将健康豚鼠30只随机分为3组,应用豚鼠全耳蜗灌流技术,分别经耳蜗灌流人工外淋巴液和不同浓度的谷氨酸2小时,记录灌流前和灌流后的耳蜗电位(CM、CAP);同时应用透射电镜技术观察灌流前后耳蜗形态学的变化。结果灌流人工外淋巴液后豚鼠耳蜗电位及形态学无改变;灌流10 mmol/L谷氨酸后CM幅度虽有下降,其非线性特点无改变,CAP阈值平均升高了35 dB;灌流20 mmol/L谷氨酸后CM幅度明显下降但仍保持其非线性特点,CAP阈值平均升高了48 dB,灌流谷氨酸后耳蜗内毛细胞及传入神经纤维出现空化。结论谷氨酸是耳蜗主要的兴奋性传入神经递质,应用外源性谷氨酸可以引起耳蜗内毛细胞及传入神经的损伤,但对耳蜗外毛细胞无影响。     

Acoustic Trauma Increases Ribbon Number and Size in Outer Hair Cells of the Mouse Cochlea

Outer hair cells (OHCs) in the mouse cochlea are contacted by up to three type II afferent boutons. On average, only half of these are postsynaptic to presynaptic ribbons. Mice of both sexes were subjected to acoustic trauma that produced a threshold shift of 44.2 ± 9.1 dB 7 days after exposure. Ribbon synapses of OHCs were quantified in post-trauma and littermate controls using immunolabeling of CtBP2. Visualization with virtual reality was used to determine 3-D cytoplasmic localization of CtBP2 puncta to the synaptic pole of OHCs. Acoustic trauma was associated with a statistically significant increase in the number of synaptic ribbons per OHC. Serial section TEM was carried out on similarly treated mice. This also showed a significant increase in the number of ribbons in post-trauma OHCs, as well as a significant increase in ribbon volume compared to ribbons in control OHCs. An increase in OHC ribbon synapses after acoustic trauma is a novel observation that has implications for OHC:type II afferent signaling. A mathematical model showed that the observed increase in OHC ribbons considered alone could produce a significant increase in action potentials among type II afferent neurons during strong acoustic stimulation.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10162-020-00777-w.     

豚鼠耳蜗外毛细胞的钾通道

目的研究外毛细胞钾离子通道的生物物理学和药理学特性.方法用细胞贴附式和内面向外式膜片钳技术,研究豚鼠耳蜗单离外毛细胞底侧膜的钾离子单通道电流.结果记录到两种钾离子单通道电流:(1)大电导型钙激活钾通道,在等渗140mmol/L KC1溶液时电导为161 26pS(内面向外式)或155±27pS(细胞贴附式),反转电位为0mV;浴液为Hanks液而电极内液为140mmo/L KC1液时,电导为133±9pS(细胞贴附式).当膜内面浴于无钙液时通道活动消失.通道呈簇状发放或快速簇状发放方式,其开启和关闭时程直方图均可用三阶指数方程拟合.双氢链霉素能够可逆性地抑制外毛细胞的大电导型钙激活钾通道,表现为电流幅度减少,在链霉素浓度增大以及细胞膜电位偏正时更明显,新霉素的抑制作用更强.(2)～44pS钾通道.结论豚鼠耳蜗外毛细胞底侧膜具有两种钾离子通道,研究了其大电导型钙激活钾通道的生物物理学和药理学特性.     

Preliminary Characterization of Voltage-Activated Whole-Cell Currents in Developing Human Vestibular Hair Cells and Calyx Afferent Terminals

We present preliminary functional data from human vestibular hair cells and primary afferent calyx terminals during fetal development. Whole-cell recordings were obtained from hair cells or calyx terminals in semi-intact cristae prepared from human fetuses aged between 11 and 18 weeks gestation (WG). During early fetal development (11–14 WG), hair cells expressed whole-cell conductances that were qualitatively similar but quantitatively smaller than those observed previously in mature rodent type II hair cells. As development progressed (15–18 WG), peak outward conductances increased in putative type II hair cells but did not reach amplitudes observed in adult human hair cells. Type I hair cells express a specific low-voltage activating conductance, G _K,L. A similar current was first observed at 15 WG but remained relatively small, even at 18 WG. The presence of a “collapsing” tail current indicates a maturing type I hair cell phenotype and suggests the presence of a surrounding calyx afferent terminal. We were also able to record from calyx afferent terminals in 15–18 WG cristae. In voltage clamp, these terminals exhibited fast inactivating inward as well as slower outward conductances, and in current clamp, discharged a single action potential during depolarizing steps. Together, these data suggest the major functional characteristics of type I and type II hair cells and calyx terminals are present by 18 WG. Our study also describes a new preparation for the functional investigation of key events that occur during maturation of human vestibular organs.     

谷氨酸／天冬氨酸转运体抗体对豚鼠听性脑干反应及毛细胞形态的影响

目的 研究谷氨酸/天冬氨酸转运体(glutamate--aspartate transporter,GLAST)抗体对豚鼠耳蜗听性脑干反应(ABR)和耳蜗毛细胞形态的影响.方法 健康豚鼠20只随机分为实验组和对照组,每组10只.实验组耳蜗鼓阶内灌注GLAST抗体,对照组灌注人工外淋巴液,观察两组术后3、6、9天ABR反应阈、耳蜗基底膜铺片和透射电镜的形态学改变.结果 实验组术后第3天ABR波形消失,术后第9天无恢复;对照组术后第3天8只动物ABR波形消失,术后第6天和第9天全部动物引出ABR波形,平均阈值分别为62.50±5.25、47.50±6.18dB SPL,差异有统计学意义(P<0.05).随着GLAST抗体灌注后时间延长,实验组内、外毛细胞及纤毛出现不同程度缺失,透射电镜显示内、外毛细胞及神经末梢胞浆、线粒体空化,细胞核染色质边集等凋亡早期征象.对照组的损伤较轻,与ABR阈值改变相一致.结论 耳蜗内GLAST抗体灌注后出现耳蜗毛细胞、神经末梢的损伤及ABR波形消失,提示GLAST抗体阻断耳蜗Corti器中的GLAST,导致谷氨酸的神经毒性表达.     

Protective Effect of Hexane and Ethanol Extract of Piper Longum L. on Gentamicin-Induced Hair Cell Loss in Neonatal Cultures

Objectives

Gentamicin (GM) is a commonly used aminoglycoside antibiotic that generates free oxygen radicals within the inner ear, which can cause vestibulo-cochlear toxicity and permanent damage to the sensory hair cells and neurons. Piper longum L. (PL) is a well-known spice and traditional medicine in Asia and Pacific islands, which has been reported to exhibit a wide spectrum of activity, including antioxidant activity. In this study, we evaluated the effect of hexane:ethanol (2:8) PL extract (subfraction of PL [SPL] extract) on GM-induced hair cell loss in basal, middle and apical regions in a neonatal cochlea cultures.

Methods

The protective effects of SPL extract were measured by phalloidin staining of cultures from postnatal day 2-3 mice with GM-induced hair cell loss. The anti-apoptosis activity of SPL extract was measured using double labeling by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and myosin-7a staining. The radical-scavenging activity of SPL extract was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay.

Results

SPL extract at a concentration of 1 µg/mL significantly inhibited GM-induced hair cell loss at basal and middle region of cochlea, while 5 µg/mL was effective against apical region hair cell loss. The protective effect of SPL extract was concentration dependent and hair cells retained their stereocilia in explants treated with SPL extract prior to treatment with 0.3 mM GM. SPL extract decreased GM-induced apoptosis of hair cells as assessed by TUNEL staining. The outer hair and inner hair counts were not decreased in SPL extract treated groups in compare to GM treated explants. Additionally, SPL extract showed concentration dependent radical scavenging activity in a DPPH assay.

Conclusion

An anti-apoptosis effect and potent radical scavenger activity of SPL extract protects from GM-induced hair cell loss at basal, middle and apical regions in neonatal cochlea cultures.     

Progression of Inner Ear Pathology in Ames Waltzer Mice and the Role of Protocadherin 15 in Hair Cell Development

The Ames waltzer (av) mouse mutant exhibits auditory and vestibular abnormalities resulting from mutation of protocadherin 15 (Pcdh15). Ames waltzer has been identified as an animal model for inner ear pathology associated with Usher syndrome type 1F. Studies correlating anatomical phenotype with severity of genetic defect in various av alleles are providing better understanding of the role played by Pcdh15 in inner ear development and of sensorineural abnormalities associated with alterations in Pcdh15 protein structure as a result of gene mutation. In this work we present new findings on inner ear pathology in four alleles of av mice with differing mutations of Pcdh15 as well as varying alterations in inner ear morphology. Two alleles with in-frame deletion mutations (Pcdh15 ^av-J and Pcdh15 ^av-2J ) and two presumptive functional null alleles (Pcdh15 ^av-3J and Pcdh15 ^av-Tg ) were studied. Light and electron microscopic observations demonstrated that the severity of cochlear and vestibular pathology in these animals correlates positively with the extent of mutation in Pcdh15 from embryonic day 18 (E18) up to 12 months. Electron microscopic analysis of immature ears indicated early abnormalities in the arrangement of stereocilia and the inner and outer hair cell cuticular plates, stereocilia rootlets, and the actin meshwork within the cuticular plate. In severe cases, displacement of the kinocilium and alterations in the shape of the cuticular plate was also observed. Mice harboring in-frame deletion mutations showed less disorganization of stereocilia and cuticular plates in the organ of Corti than the presumptive functional null alleles at P0–P10. A slower progression of pathology was also seen via light microscopy in older animals with in-frame deletions, compared to the presumptive functional null mutations. In summary, our results demonstrate that mutation in Pcdh15 affects the initial formation of stereocilia bundles with associated changes in the actin meshwork within the cuticular plate; these effects are more pronounced in the presumed null mutation compared to mutations that only affect the extracellular domain. The positive correlation of severity of effects with extent of mutation can be seen well into adulthood.     

Maintained Expression of the Planar Cell Polarity Molecule Vangl2 and Reformation of Hair Cell Orientation in the Regenerating Inner Ear

The avian inner ear possesses a remarkable ability to regenerate sensory hair cells after ototoxic injury. Regenerated hair cells possess phenotypes and innervation that are similar to those found in the undamaged ear, but little is known about the signaling pathways that guide hair cell differentiation during the regenerative process. The aim of the present study was to examine the factors that specify the orientation of hair cell stereocilia bundles during regeneration. Using organ cultures of the chick utricle, we show that hair cells are properly oriented after having regenerated entirely in vitro and that orientation is not affected by surgical removal of the striolar reversal zone. These results suggest that the orientation of regenerating stereocilia is not guided by the release of a diffusible morphogen from the striolar reversal zone but is specified locally within the regenerating sensory organ. In order to determine the nature of the reorientation cues, we examined the expression patterns of the core planar cell polarity molecule Vangl2 in the normal and regenerating utricle. We found that Vangl2 is asymmetrically expressed on cells within the sensory epithelium and that this expression pattern is maintained after ototoxic injury and throughout regeneration. Notably, treatment with a small molecule inhibitor of c-Jun-N-terminal kinase disrupted the orientation of regenerated hair cells. Both of these results are consistent with the hypothesis that noncanonical Wnt signaling guides hair cell orientation during regeneration.     

L型电压门控钙通道α1D亚基在内耳毛细胞的分布及在听力中的作用

目的探讨L型电压门控钙通道α1D亚基在内耳毛细胞的分布及在听力中的作用。方法应用免疫细胞化学荧光标记观察L型电压门控钙通道α1D亚基在牛蛙毛细胞上的分布;利用听性脑干反应(ABR)检测携带不同L型电压门控钙通道α1D基因型(α1D / 、α1D /-和α1D-/-)小鼠的听力。结果L型电压门控钙通道α1D亚基主要密集分布于毛细胞侧膜和顶膜,在毛细胞的胞核区域和纤毛丛处没有阳性荧光染色。三种α1D基因型小鼠的ABR检测结果显示,α1D / 野生型小鼠的短声反应阈正常,为34.8±5.7dBSPL;α1D /-杂合子小鼠反应阈高于同窝生α1D / 野生型小鼠,为54.4±12.4dBSPL;α1D-/-小鼠呈现全聋,ABR在100dBSPL时仍无反应。结论L型电压门控钙通道的α1D亚基分布在牛蛙毛细胞的侧膜和顶膜,在内耳的听觉生理中具有重要作用。L型电压门控钙通道α1D亚基缺失或是数量的减少均可以影响小鼠听力。     

Differential Distribution of Stem Cells in the Auditory and Vestibular Organs of the Inner Ear

The adult mammalian cochlea lacks regenerative capacity, which is the main reason for the permanence of hearing loss. Vestibular organs, in contrast, replace a small number of lost hair cells. The reason for this difference is unknown. In this work we show isolation of sphere-forming stem cells from the early postnatal organ of Corti, vestibular sensory epithelia, the spiral ganglion, and the stria vascularis. Organ of Corti and vestibular sensory epithelial stem cells give rise to cells that express multiple hair cell markers and express functional ion channels reminiscent of nascent hair cells. Spiral ganglion stem cells display features of neural stem cells and can give rise to neurons and glial cell types. We found that the ability for sphere formation in the mouse cochlea decreases about 100-fold during the second and third postnatal weeks; this decrease is substantially faster than the reduction of stem cells in vestibular organs, which maintain their stem cell population also at older ages. Coincidentally, the relative expression of developmental and progenitor cell markers in the cochlea decreases during the first 3 postnatal weeks, which is in sharp contrast to the vestibular system, where expression of progenitor cell markers remains constant or even increases during this period. Our findings indicate that the lack of regenerative capacity in the adult mammalian cochlea is either a result of an early postnatal loss of stem cells or diminishment of stem cell features of maturing cochlear cells.     

Over Half the Hair Cells in the Mouse Utricle First Appear After Birth, with Significant Numbers Originating from Early Postnatal Mitotic Production in Peripheral and Striolar Growth Zones

Many non-mammalian vertebrates produce hair cells throughout life and recover from hearing and balance deficits through regeneration. In contrast, embryonic production of hair cells declines sharply in mammals where deficits from hair cell losses are typically permanent. Hair cell density estimates recently suggested that the vestibular organs of mice continue to add hair cells after birth, so we undertook comprehensive counting in murine utricles at different ages. The counts show that 51 % of the hair cells in adults arise during the 2 weeks after birth. Immature hair cells are most common near the neonatal macula’s peripheral edge and striola, where anti-Ki-67 labels cycling nuclei in zones that appear to contain niches for supporting-cell-like stem cells. In vivo lineage tracing in a novel reporter mouse where tamoxifen-inducible supporting cell-specific Cre expression switched tdTomato fluorescence to eGFP fluorescence showed that proteolipid-protein-1-expressing supporting cells are an important source of the new hair cells. To assess the contributions of postnatal cell divisions, we gave mice an injection of BrdU or EdU on the day of birth. The labels were restricted to supporting cells 1 day later, but by 12 days, 31 % of the labeled nuclei were in myosin-VIIA-positive hair cells. Thus, hair cell populations in neonatal mouse utricles grow appreciably through two processes: the progressive differentiation of cells generated before birth and the differentiation of new cells arising from divisions of progenitors that progress through S phase soon after birth. Subsequent declines in these processes coincide with maturational changes that appear unique to mammalian supporting cells.