Multiple roles of Notch signaling in cochlear development

Notch signaling inhibits hair cell differentiation, based on studies on mice deficient in Notch signaling-related genes and its downstream genes. However, the precise mechanisms of this inhibition are unknown because it is difficult to control the timing and duration of the suppression of Notch signaling. Here, we developed a novel in vitro culture and analysis method for mouse fetal cochleae and examined the roles of Notch signaling by its reversible inhibition through the use of Notch signaling inhibitors of gamma-secretase and TNF-alpha-converting enzyme. Notch inhibition with Notch signaling inhibitor treatment increases the number of cochlear hair cells, as observed in gene deletion experiments. We elucidated that this increase is regulated by the dichotomy between hair cells and supporting cells from common progenitors. We also propose other roles of Notch signaling in cochlear development. First, Notch signaling arrests the cell cycle of the cochlear epithelium containing putative hair cells and supporting cell progenitors because Notch inhibition with inhibitor treatment increases the number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells that can differentiate into hair cells or supporting cells. Second, Notch signaling is required for the induction of Prox1-positive supporting cells. Third, Notch signaling is required for the maintenance of supporting cells.     

Forced activation of Wnt signaling alters morphogenesis and sensory organ identity in the chicken inner ear

Components of the Wnt signaling pathway are expressed in the developing inner ear. To explore their role in ear patterning, we used retroviral gene transfer to force the expression of an activated form of beta-catenin that should constitutively activate targets of the canonical Wnt signaling pathway. At embryonic day 9 (E9) and beyond, morphological defects were apparent in the otic capsule and the membranous labyrinth, including ectopic and fused sensory patches. Most notably, the basilar papilla, an auditory organ, contained infected sensory patches with a vestibular phenotype. Vestibular identity was based on: (1) stereociliary bundle morphology; (2) spacing of hair cells and supporting cells; (3) the presence of otoliths; (4) immunolabeling indicative of vestibular supporting cells; and (5) expression of Msx1, a marker of certain vestibular sensory organs. Retrovirus-mediated misexpression of Wnt3a also gave rise to ectopic vestibular patches in the cochlear duct. In situ hybridization revealed that genes for three Frizzled receptors, c-Fz1, c-Fz7, and c-Fz10, are expressed in and adjacent to sensory primordia, while Wnt4 is expressed in adjacent, nonsensory regions of the cochlear duct. We hypothesize that Wnt/beta-catenin signaling specifies otic epithelium as macular and helps to define and maintain sensory/nonsensory boundaries in the cochlear duct.     

Effects of Lipophilic Ions on Outer Hair Cell Membrane Capacitance and Motility

The outer hair cell (OHC) from the mammalian organ of Corti possesses a bell-shaped voltage-dependent capacitance function. The nonlinear capacitance reflects the activity of membrane bound voltage sensors associated with membrane motors that control OHC length. We have studied the effects of the lipophilic ions, tetraphenylborate (TPB⁻) and tetraphenylphosphonium (TPP⁺), on nonlinear capacitance and motility of isolated guinea-pig OHCs. Effects on supporting cells were also investigated. TPB⁻ produced an increase in the peak capacitance (Cm _pk ) and shifted the voltage at peak capacitance (V _pkCm ) to hyperpolarized levels. Washout reversed the effects. Perfusion of 0.4 μm TPB⁻ caused an average increase in Cm _pk of 16.3 pF and V _pkCm shift of 13.6 mV. TPP⁺, on the other hand, only shifted V _pkCm in the positive direction, with no change in Cm _pk . The contributions from native OHC and TPB⁻-induced capacitance were dissected by a double Boltzmann fitting paradigm, and by blocking native OHC capacitance. While mechanical response studies indicate little effect of TPB⁻ on the motility of OHCs which were in normal condition or treated with salicylate or gadolinium, the voltage at maximum mechanical gain (V _{δ Lmax} ) was shifted in correspondence with native V _pkCm , and both changed in a concentration-dependent manner. Both TPB⁻-induced changes in Cm _pk and V _pkCm were affected by voltage prepulses and intracellular turgor pressure. TPB⁻ induced a voltage-dependent capacitance in supporting cells whose characteristics were similar to those of the OHC, but no indication of mechanical responses was noted. Our results indicate that OHC mechanical responses are not simply related to quantity of nonspecific nonlinear charge moved within the membrane, but to the effects of motility voltage-sensor charge movement functionally coupled to a mechanical effector. Received: 14 May 1998/Revised: 24 August 1998     

双重荧光染色监测听毛细胞游离钙

豚鼠耳蜗分离外毛细胞经fluo-3和fura-red染色后,用激光扫描共聚焦显微镜监测其胞内游离钙的空间分布和动态变化,以fluo-3/fura-red荧光比值大小指示游离钙浓度的高低。外毛细胞胞内游离钙呈不均匀分布,荧光比值分别为细胞质1.71±0.85,质膜1.61±0.75,表皮板1.47±0.65及胞核1.39±0.66,显示细胞质游离钙浓度最高而胞核游离钙浓度最低。静息状态下连续扫描时荧光比值变化小于0.1,而在受到机械刺激后荧光比值增加幅度达0.3—1.2。实验结果表明,fluo-3和fura-red双发射比例法能更准确反映听毛细胞胞内钙的空间分布和动态变化,第二信使钙可能参与了听毛细胞的机械-电换能过程,研究了换能过程中听毛细胞钙信号的时空模式。     

Mitochondrial Syndromic Sensorineural Hearing Loss

Mitochondrial diseases (MD) are a clinically heterogeneous group of disorders that arise as a result of dysfunction of the mitochondrial respiratory chain. Sensorineural hearing loss (SNHL) is often associated to mitochondrial dysfunctions both in syndromic, nonsyndromic forms. SNHL has been described in association to different mitochondrial multisystemic syndromes, often characterized by an important neuromuscular involvement. Because of the clinical relevance of the associated neurological symptoms, the occurrence of SNHL is often underestimated and undiagnosed. In this study we evaluated the incidence of SNHL in a group of 17 patients with MD. We detected some degree of hearing impairment in 8/17 patients (47%), thus confirming the frequency of hearing impairment in MD. Furthermore, we want to highlight the role of the audiologist and otolaryngologist in the diagnosis and characterization of a MD, which should be suspected in all the cases in which the hearing loss is associated to signs and symptoms characteristic of mitochondrial dysfunction, especially if the family history is positive for hearing loss or MD in the maternal line.     

Malformation of stria vascularis in the developing inner ear of the German waltzing guinea pig

Auditory function and cochlear morphology have previously been described in the postnatal German waltzing guinea pig, a strain with recessive deafness. In the present study, cochlear histopathology was further investigated in the inner ear of the developing German waltzing guinea pig (gw/gw). The lumen of the cochlear duct diminished progressively from embryonic day (E) 35 to E45 and was absent at E50 because of the complete collapse of Reissner's membrane onto the hearing organ. The embryonic stria vascularis, consisting of a simple epithelium, failed to transform into the complex trilaminar tissue seen in normal animals and displayed signs of degeneration. Subsequent degeneration of the sensory epithelium was observed from E50 and onwards. Defective and insufficient numbers of melanocytes were observed in the developing gw/gw stria vascularis. A gene involved in cochlear melanocyte development, Pax3, was markedly reduced in lateral wall tissue of the cochlea of both E40 and adult gw/gw individuals, whereas its expression was normal in the skin and diaphragm muscle of adult gw/gw animals. The Pax3 gene may thus be involved in the pathological process but is unlikely to be the primary mutated gene in the German waltzing guinea pig. TUNEL assay showed no signs of apoptotic cell death in the developing stria vascularis of this type of guinea pig. Thus, malformation of the stria vascularis appears to be the primary defect in the inner ear of the German waltzing guinea pig. Defective and insufficient numbers of melanocytes might migrate to the developing stria vascularis but fail to provide the proper support for the subsequent development of marginal and basal cells, thereby leading to stria vascularis malformation and dysfunction in the inner ear of the German waltzing guinea pig.     

Prolonged maturation of cochlear function in the barn owl after hatching

Cochlear microphonics (CMs), which represent the electrical activity of hair cells, and compound action potentials (CAPs), which represent the activity of the auditory nerve, were recorded from the round window of the inner ear, in owlets aged between 5 and 97 days posthatching, i.e., from soon after hatching to beyond fledgling. At the earliest ages examined, animals showed very insensitive CM and virtually no CAP responses. Thus, hearing in barn owls develops entirely posthatching and the birds appear to be profoundly deaf well into the second week. Thresholds improved gradually after that and CMs reached their adult sensitivity at 5 weeks posthatching at all frequencies. Compound action potential responses appeared progressively later with increasing frequency. Adult neural sensitivity was achieved about 1 week later than for the CM responses at most frequencies, but took until 9–10 weeks posthatching at the highest frequencies (8–10 kHz). This indicates an apex-to-base maturation sequence of neural sensitivity within the cochlea, with a disproportionately long period to maturity for the most basal regions. Compound action potential amplitudes matured even later, at about 3 months posthatching, at all frequencies. This suggests a prolonged immaturity in the temporal synchrony of spiking in the auditory nerve.     

Hair Cells – Beyond the Transducer

Overview This review considers the “tween twixt and twain” of hair cell physiology, specifically the signaling elements and membrane conductances which underpin forward and reverse transduction at the input stage of hair cell function and neurotransmitter release at the output stage. Other sections of this review series outline the advances which have been made in understanding the molecular physiology of mechanoelectrical transduction and outer hair cell electromotility. Here we outline the contributions of a considerable array of ion channels and receptor signaling pathways that define the biophysical status of the sensory hair cells, contributing to hair cell development and subsequently defining the operational condition of the hair cells across the broad dynamic range of physiological function.     

内耳形态学和生理指标对缺铁性肾虚耳聋大鼠模型的评价作用

目的探讨耳蜗显微结构和生理指标对缺铁性肾虚耳聋大鼠模型的评价作用。方法选用体重30～32 g、无耳疾、听性脑干反应(auditory brainstem response,ABR)阈值正常的1～2周龄SPF级SD大鼠120只,雌雄分养,分为缺铁组80只、正常对照组40只,饲养时间12周;以出现肾虚证和至少一耳ABR阈值≥15 dB,作为判断肾虚耳聋的标准,获得缺铁性肾虚耳聋大鼠22只,从中选取肾虚耳聋大鼠20只,同时以20只正常大鼠作对照。观察耳蜗血管纹、螺旋器等耳蜗显微结构变化,检测ABR阈值以及血红蛋白和血清铁等指标的变化。结果实验组和正常对照大鼠的血红蛋白和血清铁分别为11.80 g/L,4.5μmol/L和45.9 g/L,22.23μmol/L,ABR阈值分别为(30±5)dB和(10±5)dB;实验组血管纹血管明显减少;螺旋器毛细胞听毛有缺失、倒伏现象。结论缺铁性肾虚耳聋大鼠血红蛋白、血清铁和ABR阈值,以及耳蜗血管纹、螺旋器等耳蜗显微结构变化等指标,均较为稳定,是较好的评价指标。     

5-HT<Subscript>6/7</Subscript> Receptor Antagonists Facilitate Dopamine Release in the Cochlea via a GABAergic Disinhibitory Mechanism

In humans, serotonin (5-HT) has been implicated in numerous physiological and pathological processes in the peripheral auditory system. Dopamine (DA), another transmitter of the lateral olivocochlear (LOC) efferents making synapses on cochlear nerve dendrites, controls auditory nerve activation and protects the sensory nerve against overactivation. Using in vitro microvolume superfusion techniques we tested 5-HT₆ and 5-HT₇ receptor antagonists whether they can influence dopamine (DA) release from the guinea-pig cochlea in control and in ischemic conditions using currently available and new 5-HT₆ and 5-HT₇ antagonists and mixed antagonists, which were synthesized and characterized for the current study. While the 5-HT₇ antagonist SB-258719 was ineffective, SB-271046, which blocks the 5-HT₆ receptor, caused a significant increase in cochlear DA release what is contradictory with the excitatory nature of this type of receptor. Moreover, the mixed 5-HT_6/7 antagonist EGIS-12233 induced an even more pronounced increase in the resting DA release. To understand why the block of an excitatory receptor results in an increase instead of a decrease in function, we investigated the possible involvement of an indirect neural mechanism through an inhibitory system. In the presence of the GABA_A receptor blocker bicuculline, EGIS-12233 failed to increase the release of DA, suggesting that the serotonin receptor modulation of DA release from the lateral olivocochlear efferents in the cochlea was produced indirectly by decreasing the GABAergic inhibitory tone on dopaminergic nerve endings. The mixed 5-HT₇/D₄ receptor antagonist EGIS-11983 significantly increased both the stimulation-evoked and the resting DA release, while the selective D4 blocker L-741,741 alone had no significant effect. Ischemia, simulated by oxygen and glucose deprivation from the perfusion solution had no action on the effect of the drugs. Drugs that can increase the release of DA from LOC terminals in the cochlea may have a role in the treatment of sensorineural hearing loss.