Notch signalling pathway mediates hair cell development in mammalian cochlea.

The mammalian cochlea contains an invariant mosaic of sensory hair cells and non-sensory supporting cells reminiscent of invertebrate structures such as the compound eye in Drosophila melanogaster. The sensory epithelium in the mammalian cochlea (the organ of Corti) contains four rows of mechanosensory hair cells: a single row of inner hair cells and three rows of outer hair cells. Each hair cell is separated from the next by an interceding supporting cell, forming an invariant and alternating mosaic that extends the length of the cochlear duct. Previous results suggest that determination of cell fates in the cochlear mosaic occurs via inhibitory interactions between adjacent progenitor cells (lateral inhibition). Cells populating the cochlear epithelium appear to constitute a developmental equivalence group in which developing hair cells suppress differentiation in their immediate neighbours through lateral inhibition. These interactions may be mediated through the Notch signalling pathway, a molecular mechanism that is involved in the determination of a variety of cell fates. Here we show that genes encoding the receptor protein Notch1 and its ligand, Jagged 2, are expressed in alternating cell types in the developing sensory epithelium. In addition, genetic deletion of Jag2 results in a significant increase in sensory hair cells, presumably as a result of a decrease in Notch activation. These results provide direct evidence for Notch-mediated lateral inhibition in a mammalian system and support a role for Notch in the development of the cochlear mosaic.

Persistence of Ca(v)1.3 Ca2+ channels in mature outer hair cells supports outer hair cell afferent signaling.

Outer hair cells (OHCs) are innervated by type II afferent fibers of as yet unknown function. It is still a matter of debate whether OHCs perform exocytosis. If so, they would require presynaptic Ca2+ channels at their basal poles where the type II fibers make contacts. Here we show that L-type Ca2+ channel currents (charge carrier, 10 mM Ba2+) present in neonatal OHCs [postnatal day 1 (P1) to P7] decreased from approximately 170 to approximately 50 pA at approximately the onset of hearing. Ba2+ currents could hardly be measured in mature mouse OHCs because of their high fragility, whereas in the rat, the average Ba2+ current amplitude of apical OHCs was 58 +/- 9 pA (n = 20, P19-P30) compared with that of the inner hair cells (IHCs) of 181 +/- 50 pA (n = 24, P17-P30). Properties of Ba2+ currents of mature OHCs resembled those of neonatal OHCs. One exception was the voltage dependence of activation that shifted between birth and P12 by +9 mV toward positive voltages in OHCs, whereas it remained constant in the IHCs. Ca(v)1.3-specific mRNA was detected in mature OHCs using cell-specific reverse transcription (RT)-PCR and in situ hybridization. Ca(v)1.3 protein was stained exclusively at the base of mature OHCs, in colocalization with the ribbon synapse protein CtBP2 (C-terminal binding protein 2)/RIBEYE. When current sizes were normalized to the estimated number of afferent fibers or presynaptic ribbons, comparable values for IHCs and OHCs were obtained, a finding that together with the colocalization of Ca(v)1.3 and CtBP2/RIBEYE protein strongly suggests a role for Ca(v)1.3 channels in exocytosis of mature OHCs.

Development and properties of stereociliary link types in hair cells of the mouse cochlea.

The hair bundles of outer hair cells in the mature mouse cochlea possess three distinct cell-surface specializations: tip links, horizontal top connectors, and tectorial membrane attachment crowns. Electron microscopy was used to study the appearance and maturation of these link types and examine additional structures transiently associated with the developing hair bundle. At embryonic day 17.5 (E17.5), the stereocilia are interconnected by fine lateral links and have punctate elements distributed over their surface. Oblique tip links are also seen at this stage. By postnatal day 2 (P2), outer hair cell bundles have a dense cell coat, but have lost many of the lateral links seen at E17.5. At P2, ankle links appear around the base of the bundle and tectorial membrane attachment crowns are seen at the stereociliary tips. Ankle links become less apparent by P9 and are completely lost by P12. The appearance of horizontal top connectors, which persist into adulthood, occurs concomitant with this loss of ankle links. Treatment with the calcium chelator BAPTA or the protease subtilisin enabled these links to be further distinguished. Ankle links are susceptible to both treatments, tip links are only sensitive to BAPTA, and tectorial membrane attachment crowns are removed by subtilisin but not BAPTA. The cell-coat material is partially sensitive to subtilisin alone, while horizontal top connectors resist both treatments. These results indicate there is a rich, rapidly changing array of different links covering the developing hair bundle that becomes progressively refined to generate the mature complement by P19.

Expression of prestin mRNA in the organotypic culture of rat cochlea.

To quantitate in absolute terms the prestin mRNA levels in the explant culture of rat cochlea, we used competitive RT-PCR with a synthetic internal cRNA standard. Prestin gene expression was found at levels of 100 fg specific mRNA/microg total RNA on postnatal day 3, which corresponds to about 300 copies per outer hair cell (OHC) and is indicative of an intermediate level of expression. Two days of culturing resulted in an increase of prestin mRNA levels and in the formation of an apical-basal gradient (p<0.001). To elucidate the variations the prestin mRNA levels undergo as a result of damage to the organ of Corti, we exposed the explant cultures to ischemia and hypoxia. While total RNA was observed to remain unchanged, the numbers of OHCs and the prestin mRNA levels were found to decrease by about 20% and 35%, respectively, compared to normoxia. In conclusion, we showed that the prestin mRNA levels during in vitro development increase and form an apical-basal gradient within 2 days in culture, similar to the postnatal in vivo development. Hypoxia and ischemia result in a decrease of the prestin mRNA level in parallel with OHC loss. The prestin mRNA level can therefore be used as marker of damage to or loss of OHCs.

Distribution of actin and tubulin in outer hair cells isolated from developing rat cochlea: a quantitative study.

In the organ of Corti, outer hair cells (OHCs) are sensory effectors responsible for the high sensitivity and sharp tuning of the cochlea. Whilst the distribution and organization of actin and tubulin in adult OHCs have been extensively studied, less is known about developing OHCs. In this study we use a quantitative cytometric approach on rat isolated OHCs to measure the distribution of these cytoskeletal proteins from the first stages of development (postnatal day 5) to the adult stage. We report a general decrease in both actin and tubulin concentrations during OHC maturation. Actin first decreases in the apical domain, and then in the medio-basal domain. In the apical domain, this could be related to the physiological reduction in the number of stereocilia occurring during ciliogenesis. In the medio-basal domain, the decrease, accompanied by a redistribution of actin toward the lateral wall, is possibly related to the general reorganization of cytoplasmic organelles, to the maturation of the cortical lattice, and to cell growth. Tubulin concentration decreases regularly in both the apical and the medio-basal domains. This developmental change in tubulin concentration could be due to the regression of the kinocilium that occurs by the end of ciliogenesis, and, as argued for actin, to the general reorganization of cytoplasmic organelles, and to cell growth.

Immunohistochemical localization of adenosine 5'-triphosphate-gated ion channel P2X(2) receptor subunits in adult and developing rat cochlea.

Substantial in vitro and in vivo data support a role for extracellular adenosine 5;-triphosphate (ATP) and associated P2 receptors in cochlear function. However, the precise spatiotemporal distribution of the involved receptor protein(s) has not been determined. By using a specific antiserum and immunoperoxidase labeling, the tissue distribution of the P2X(2) subunit of the ATP-gated ion channel was investigated. Here, we describe the first extensive immunohistochemical mapping of P2X(2) receptor subunits in the adult and developing rat cochlea. In the adult, immunoreactivity was observed in most cells bordering on the endolymphatic compartment (scala media), particularly in the supporting cells. Hair cells were not immunostained by the P2X(2) antiserum, except for outer hair cell stereocilia. In addition, weak immunolabeling was observed in some spiral ganglion neurons. P2X(2) receptor subunit protein expression during labyrinthine ontogeny was detected first on embryonic day 19 in the spiral ganglion and in associated nerve fibers extending to the inner hair cells. Immunostaining also was observed underneath outer hair cells, and, by postnatal day 6 (P6), intense immunolabeling was seen in the synaptic regions of both types of hair cell. Supporting cells of the sensory epithelium were labeled at P0. This labeling became most prominent from the onset of cochlear function (P8-P12). Conversely, expression in the vascular stria declined from this time. By P21, the pattern of immunolabeling was similar to that found in the adult. The localization and timing of P2X(2) immunoreactivity suggest involvement of extracellular ATP and associated ATP-gated ion channels in important physiological events, such as inner ear ontogeny, sound transduction, cochlear micromechanics, electrochemical homeostasis, and auditory neurotransmission.

Afferent innervation of outer and inner hair cells is normal in neonatally de-efferented cats.

It has been hypothesized that normal pruning of exuberant branching of afferent neurons in the developing cochlea is caused by the arrival of the olivocochlear efferent neurons and the resulting competition for synaptic sites on hair cells. This hypothesis was supported by a report that afferent innervation density on mature outer hair cells (OHCs) is elevated in animals deefferented at birth, before the olivocochlear system reaches the outer hair cell area (Pujol and Carlier [1982] Dev. Brain Res. 3:151-154). In the current study, this claim was evaluated quantitatively at the electron microscopic level in four cats that were de-efferented at birth and allowed to survive for 6-11 months. A semiserial section analysis of 156 OHCs from de-efferented and normal ears showed that, although de-efferentation essentially was complete in all four cases, the number and distribution of afferent terminals on OHCs was indistinguishable from normal, and the morphology of afferent synapses was normal in both the inner hair cell area and the OHC area. Thus, the postnatal presence of an efferent system is not required for the normal development of cochlear afferent innervation, and the synaptic competition hypothesis is not supported.

Expression of the mouse Macf2 gene during inner ear development.

Plakins, a family of linker proteins that connect cytoskeletal elements to cellular junctions and the extracellular matrix, are primarily responsible for the mechanical properties of cells and tissues. They include desmoplakin, envoplakin, plectin, dystonin/BPAG1, and Kakapo. Mutations in plakins cause several skin, muscular and neurological disorders. Macrophins are a recently discovered subfamily of plakins with binding domains for actin, intermediate filaments and microtubules. Characteristic features of macrophins include variable actin binding domains, a central rod domain containing both plectin and spectrin repeats, and a C-terminus containing EF hands and GAS2/GAR22 domain. We have examined expression of mouse Macf2, encoding macrophin-2, in adult tissues and in the developing, neonatal, and mature inner ear by in situ hybridization. Northern blot analysis identified three large tissue-specific Macf2 transcripts: a 16-kb mRNA in skeletal muscle and heart, a 15-kb mRNA in brain, and a 9-kb mRNA in RNA from ovary plus uterus. In situ hybridization of the developing mouse inner ear indicated that Macf2 is expressed in the otocyst at day 12.5, in the sensory epithelium by embryonic day 16.5, and in both inner and outer hair cells by day 16.5. Macf2 is expressed in the bodies of both sensory and motor neurons in the central and peripheral nervous system, including the auditory pathway. The Macf2 protein could be involved in the regulation of cytoskeletal connections to cellular junctions and play an important structural role in organs, such as the inner ear, that are subjected to strong mechanical forces.

Expression of potassium channels in gerbil outer hair cells during development does not require neural induction.

Mammalian outer hair cells (OHCs) contain Ca and K channels in their synaptic pole. We questioned if the ontogeny of potassium currents of OHCs depends on the neural induction of early afferent contact. By recording whole-cell currents of OHCs grown in organotypic cultures deprived of afferent innervation, we show that a Ca-activated K channel is expressed in these cells, suggesting that the ontogeny of the K channel is an intrinsic process.

Developmental mRNA expression of the alpha10 nicotinic acetylcholine receptor subunit in the rat cochlea.

A recently discovered alpha10 subunit of the nicotinic acetylcholine receptor (nAChR) family is believed to form a heteromeric receptor with the alpha9 nAChR subunit in auditory hair cells. In the present study, the alpha10 nAChR subunit expression in the developing and adult rat inner ear was analyzed by PCR and localized using isotopic in situ hybridization. Unlike the alpha9 subunit, the alpha10 subunit was not detected at embryonic day 18 (E18). From E21 through postnatal day 15 (P15), the alpha10 subunit was localized over both inner hair cell (IHC) and outer hair cell (OHC) regions, but in the mature cochlea detectable levels of alpha10 mRNA were found only over the OHC region. From E21 through adult ages, there was also a small but consistent basal to apical gradient of alpha10 expression; that is, higher levels in basal regions and lower levels in apical regions. Previously, we detected the alpha9 nAChR subunit over IHCs as early as E18 and throughout adult ages with a clear basal-apical gradient of expression. Our studies raise the question of whether the alpha9 and alpha10 subunits are differentially regulated during embryonic and postnatal development.

Choline-acetyltransferase-like immunoreactivity in the organ of Corti of the rat during postnatal development.

The mammalian cochlea receives efferent innervation from neurons located in the superior olivary complex. This efferent olivocochlear innervation is divided in two separate systems, lateral and medial, which mainly innervate afferent dendrites connected to inner hair cells and the cell body of outer hair cells, respectively. Besides other substances, lateral and medial efferent terminals of the adult cochlea use acetylcholine (ACh) as a neurotransmitter. In this study, we have used immunocytochemistry to detect the presence of choline acetyltransferase (ChAT), the synthesizing enzyme of ACh, in efferent olivocochlear terminals during the development of the rat. The appearance and distribution of immunoreactivity to ChAT has been studied in developing rat cochleas from birth (postnatal day 1, P1) to adulthood. Attention was paid to the temporal relationships between the expression of ChAT, the presence of other putative neuroactive substances, the onset of hearing and other developmental phenomena. Our results indicate that ChAT-like immunoreactivity is already present at birth (P1) in the region of inner hair cells, that it appears at P3 in the outer hair cell area and that it reaches an adult pattern of distribution by P15. ACh may thus be present early in the developing cochlea, before the onset of hearing, as it also occurs with other putative transmitters/modulators such as enkephalins, CGRP or GABA. It is suggested that ACh could be involved in the modulation of sound-evoked potentials as soon as they appear, and in the regulation of other developmental phenomena such as neurite outgrowth or synaptogenesis.

Synaptophysin and GAP-43 proteins in efferent fibers of the inner ear during postnatal development.

A rearrangement of afferent and efferent fibers occurs in the postnatal development of the inner ear. Growth and synaptogenesis was explored during this critical period by immunohistochemically monitoring the expression of GAP-43 and synaptophysin. Both proteins were colocalized in efferent fibers beyond postnatal day 3 (pn3). Two distinct synaptophysin- and GAP-43-positive fibers innervated different parts of inner hair cells in the first and second postnatal weeks, respectively. GAP-43-positive efferents projecting to outer hair cells upregulated synaptophysin with base to apex gradient between postnatal day 5 and postnatal day 14. In efferents projecting to outer hair cells GAP-43 was downregulated about 6 days beyond synaptogenesis. In efferents projecting to inner hair cells, however, GAP-43 remained upregulated even beyond pn18, indicating continuous synapse replacement of this fiber type. Both proteins thus improved as excellent markers for growth and synaptogenesis of distinct postnatal efferent fibers.

Ontogenesis of rat cochlea. A quantitative study of the organ of Corti.

A systematic quantitative set of data concerning the organ of Corti in developing Sprague-Dawley rats at intervals from 18 days of gestation to 10 days after birth (DAB) is provided in this study. Using phalloidin staining, the total number of inner and outer hair cells, the whole length of cochlea, as well as the diameter of inner and outer hair cells and the intercellular space between inner hair cells were determined in order to analyze the quantitative change of inner and outer hair cells during development and to explore some roles of the factors regulating the growth of cochlea. The results show that: (1) The length of cochlea approached its adult size by 7DAB. (2) The growth of the extreme part of the apex was responsible for the delayed elongation of the cochlea. (3) Growth in the cochlear length mainly results from an increase of cell diameter tempered by a decrease of intercellular space. (4) The adult size of inner and outer hair cells was obtained by 7-14DAB. (5) The final number of inner and outer hair cells was reached at 3DAB and remained constant through adulthood. No significant hair cell overproduction and cell death were observed during ontogenesis of the cochlea. The negligible importance of overproduction and missing hair cells during hair cell differentiation suggest that there is a precise regulation phenomenon for producing the right spatial organization of the organ of Corti.

Development of cochlear active mechanisms in humans differs between gender.

Despite onset of function early during the third term of gestation, the human auditory system demonstrates continued maturation, thought previously to occur primarily at the neural level. The electromotile properties of outer hair cells appear to contribute substantially to hearing sensitivity and frequency selectivity and lead to the generation of otoacoustic emissions (OAEs). This report demonstrates continued development of cochlear active mechanisms (i.e. end-organ level) after onset of cochlear function, as reflected by OAEs. Significant gender differences also are reported, corresponding to recently observed intersex differences in cochlear length and precursory to gender differences observed in the adult.

Distribution of actin in developing outer hair cells in the gerbil.

During the two weeks following the onset of cochlear function in the gerbil, active cochlear processes appear to mature. The active cochlear processes likely involve outer hair cells with their specialized lateral wall structures, including the subsurface cisternae and associated cytoskeletal elements. We have previously demonstrated that the subsurface cisternae mature gradually during the time that active cochlear processes mature in the gerbil. In the study reported here, we used postembedding immunocytochemical electron microscopy to investigate whether actin labelling associated with the cortical cytoskeleton of the gerbil outer hair cell increased concomitantly. In contrast to the gradual development of the subsurface cisternae, actin labelling in the region of the cortical cytoskeleton significantly increased during the onset of cochlear function and maintained this level during the time that active cochlear processes mature. Thus, it appears that increased actin adjacent to the lateral plasma membrane of the outer hair cell is related to the onset of cochlear function rather than to the maturation of active cochlear processes.

A developmental study of distortion product otoacoustic emission (2f1-f2) suppression in humans.

Suppression of the 2f1-f2 distortion product otoacoustic emission (DPOAE) provides an effective paradigm for the study of functional cochlear maturation in humans. DPOAE iso-suppression tuning curves (STCs) represent some aspect of peripheral filtering, probably related to the boundaries of distortion generation. Studies conducted thus far suggest that the cochlear tuning assessed by this technique is adult-like in humans by term birth (Abdala et al., Hear. Res. 98 (1996) 38-53; Abdala and Sininger, Ear Hear. 17 (1996) 374-385). However, there have been no studies of cochlear tuning in premature human neonates. DPOAE STCs and suppression growth functions were measured from 14 normal-hearing adults, 33 term and 85 premature neonates to investigate the developmental time course of cochlear frequency resolution and non-linearity. Premature neonates showed non-adult-like DPOAE suppression at f2 of 1500 and 6000 Hz: (1) STCs were narrower in width (Q10) and steeper in slope on the low-frequency flank of the tuning curve; (2) suppressor tones lower in frequency than f2 produced atypically shallow growth of DPOAE suppression. The influence of immature conductive pathways cannot be entirely ruled out as a factor contributing to these results. However, findings may indicate that an immaturity exists in cochlear frequency resolution and non-linearity just prior to term birth. The bases of this immaturity are hypothesized to be outer hair cell in origin.

Postnatal development of membrane specialisations of gerbil outer hair cells.

Using a combination of freeze-fracture and thin sections, this study examines the maturation of the membrane specialisations of the gerbil outer hair cells (OHC) between 2 and 16 days after birth (DAB). The apical membrane, the junctional region around the neck of the cell, and the lateral and basal membranes are described. The results suggest a sequential development of the different components of the lateral wall. Intramembrane protein particles (IMP), the putative OHC motor elements, were found to be present at low density at 2 DAB and increased in density from 2200 IMP/microns 2 at 2 DAB to 4131/ microns 2 at 8 DAB. OHCs have been reported as showing electromotility from 8 DAB onward. IMPs continue to increase in density until mature values are attained at 16 DAB. Sub-surface cisternae did not appear until 8 DAB, with a single layer being complete by 10 DAB. Pillar structures, proposed to be related to the cytoskeletal lattice, first appear at 10 DAB. The apical membrane of the immature hair cell is characterised by the presence of pits related to the endocytosis of vesicles, and tip-links between stereocilia, thought to be associated with sites of ion channel opening, are present at 2 DAB. The junctional region comprises two areas which mature at differing rates: an apical-most region which attains an adult-like appearance by 8 DAB and a basal-ward region which continues to increase in complexity until mature at 16 DAB. The functional significance of the results are discussed in relation to the possible roles of the junctional regions and the proposed sites of the OHC motor elements.

Postnatal development of the organ of Corti in cats: a light microscopic morphometric study.

This study quantitatively characterizes the development of the major morphological features of the organ of Corti during the first 2 weeks postnatal, the period when the cat auditory system makes the transition from being essentially non-functional to having nearly adult-like responses. Four groups of kittens (n = 3) were studied at one day postnatal (P1), P5, P10, P15, and compared to adults. Measurements were made of the organ of Corti at 3 cochlear locations: 20%, 60% and 85% of basilar membrane length from the base cochlear locations which in the adult correspond to best frequencies of approximately 20 kHz, 2 kHz and 500 Hz, respectively. In addition, measurements of basilar membrane length and opening of the tunnel of Corti were made in 20 cochlear specimens from kittens aged P0-P6. Results indicate that: (i) at P0 the basilar membrane has attained adult length, and the tunnel of Corti is open over approximately the basal one-half of the cochlea; (ii) the initial opening of the tunnel of Corti occurs at a site about 4 mm from the cochlear base (best frequency of approximately 25 kHz in the adult cochlea); (iii) the thickness of the tympanic cell layer decreases markedly at the basal 20-kHz location; (iv) the areas of the tunnel of Corti and space of Nuel and the angulation of the inner hair cells (IHC) relative to the basilar membrane all show marked postnatal increases at both the middle and apical locations; (v) IHC are nearly adult-like in length and shape at birth, whereas the OHC (at 2-kHz and 500-Hz locations) undergo marked postnatal changes; (vi) disappearance of the marginal pillars and maturation of the supporting cells are not yet complete by P15.

Alteration of 6-phosphofructo-1-kinase subunits during neonatal maturation of the rat cochlear cells.

During postnatal development of rat cochlear cells and the onset of hearing (10-23 days), the increasing endocochlear potential and energy requirements are largely provided by increased glucose utilization. It is well established that the ability of maturing rat tissues to use glucose is directly related to alteration of 6-phosphofructo-1-kinase (PFK) subunits. To gain insight into the alteration of PFK subunit levels in the cochlea from 6 to 60 days of age, PFK subunit types were measured in sections of paraffin-embedded temporal bone using IgG specific for each type of PFK subunit and quantified by computer image analysis. Although the L-type and C-type subunits did not exhibit statistically significant changes in the cochlear structures during maturation, the levels of M-type subunit in the stria vascularis cells, spiral ligament cell types I, II, and III, outer hair cells, inner hair cells, and support cells significantly increased. Also, the type IV and V spiral ligament fibrocytes during this period did not exhibit significant alterations of the M-type subunit. These data suggest that during neonatal development of the cochlear, the elevated levels of the M-type subunit are associated with increased glucose utilization and the onset of hearing.

Lateral wall protein content mediates alterations in cochlear outer hair cell mechanics before and after hearing onset.

Specialized outer hair cells (OHCs) housed within the mammalian cochlea exhibit active, nonlinear, mechanical responses to auditory stimulation termed electromotility. The extraordinary frequency resolution capacity of the cochlea requires an exquisitely equilibrated mechanical system of sensory and supporting cells. OHC electromotile length change, stiffness, and force generation are responsible for a 100-fold increase in hearing sensitivity by augmenting vibrational input to non-motile sensory inner hair cells. Characterization of OHC mechanics is crucial for understanding and ultimately preventing permanent functional deficits due to overstimulation or as a consequence of various cochlear pathologies. The OHCs' major structural assembly is a highly-specialized lateral wall. The lateral wall consists of three structures; a plasma membrane highly-enriched with the motor-protein prestin, an actin-spectrin cortical lattice, and one or more layers of subsurface cisternae. Technical difficulties in independently manipulating each lateral wall constituent have constrained previous attempts to analyze the determinants of OHCs' mechanical properties. Temporal separations in the accumulation of each lateral wall constituent during postnatal development permit associations between lateral wall structure and OHC mechanics. We compared developing and adult gerbil OHC axial stiffness using calibrated glass fibers. Alterations in each lateral wall component and OHC stiffness were correlated as a function of age. Reduced F-actin labeling was correlated with reduced OHC stiffness before hearing onset. Prestin incorporation into the PM was correlated with increased OHC stiffness at hearing onset. Our data indicate lateral wall F-actin and prestin are the primary determinants of OHC mechanical properties before and after hearing onset, respectively.