The inner ear of the lungfish Protopterus

The sensory end organs of the inner ear of the lungfish, Protopterus, were examined using scanning and transmission electron microscopy. The utricle has a structure and hair cell orientation pattern that are typical for vertebrates, although the hair cells are unusually large. There are the typical three semicircular canals extending from the utricle, with the typical hair cell orientations, but the lateral canal sensory crista looks like the "hemicrista" of some amphibians and amniotes, lacking a saddle-shaped flare on one wall of the ampulla. Unlike most vertebrates that have the saccule and lagena as two separate pouches ventral to the utricle, the lungfish has a single large ventral pouch that contains a single large pasty otoconial mass. This mass covers two hair cell patches, each like a striola with prominent hair cell ciliary bundles, that are presumed to represent saccular and lagenar maculae. However, these two major sensory patches are not completely separate maculae because they lie within a less densely populated field of smaller hair cells, which forms an extrastriolar region that surrounds and fills the region between the two striolae of higher hair cell density. The more caudal lagenar striola is a vertically elongated stripe with hair cell orientation vectors facing antiparallel on either side of a midline drawn vertically along the macula, resembling the macula lagena of some bony fishes but not of tetrapods. The more rostral saccular striola is a curving band with hair cell orientation vectors facing away from its midline, but because this macula curves in three dimensions, the vectors at the rostral end of this striola are oriented mediolaterally, whereas the vectors on the caudal half of this striola are oriented dorsoventrally. The presence of a macula neglecta was confirmed near the posterior canal as a tiny single patch of a few dozen hair cells with all the cell orientations directed caudally. The ciliary bundles on the cells in the striolar-like regions of all of three otolithic organs average over 80 cilia, a number far greater than for any other fish studied to date. The features of the single sacculolagenar pouch with separate striolar-like regions, the cellular orientation in the otolith organs, and the large cells and ciliary bundles in Protopterus also were observed in specimens of the other extant lungfish genera, Lepidosiren and Neoceratodus.     

Glial fibrillary acidic protein expression and promoter activity in the inner ear of developing and adult mice.

The intermediate filament glial fibrillary acidic protein (GFAP) is a classic marker for several types of glial cells, including astrocytes and nonmyelinating Schwann cells. The pattern of expression of GFAP in the postnatal murine inner ear, from postnatal day 3 (P3) to P38, was studied by anti-GFAP immunostaining in wild-type mice as well as in two lines of transgenic mice expressing either beta-galactosidase (LacZ) or green fluorescent protein (GFP) under the control of the GFAP promoter. Analysis of protein and promoter activity shows that several classes of supporting cells in the sensory epithelia, as well as Schwann cells and satellite cells express GFAP. Early after birth, all cochlear supporting cells express GFAP, in a gradient decreasing in intensity from base to apex. After P15, GFAP expression in the organ of Corti is mostly restricted to supporting cells of the inner hair cell area (i.e., inner border and inner phalangeal cells) and outer hair cell area (i.e., Deiters' cells). A small population of limbic cells also showed expression in a base-to-apex gradient. In the vestibular organs, high expression was detected in supporting cells in extrastriolar regions of the utricular macula and in the canal ampullae, with weaker staining in the saccular macula. These results suggest that supporting cells of the inner ear have important similarities to glial cells and may play roles similar to those of astrocytes or Schwann cells in supporting the normal development and maintenance of neurons and sensory cells of the inner ear.     

Complementary and layered expression of Ephs and ephrins in developing mouse inner ear

The distributions of the Eph-class receptors EphA4 and EphB1, and their ligands ephrin-A2, ephrin-B1, and ephrin-B2, were analysed by immunostaining in the mouse inner ear. Complementary patterns of EphA4 and its potential ligand ephrin-A2 were found, with ephrin-A2 in many of the structures lining the cochlear duct and within the cochlear nerve cells, and EphA4 in the deeper structures underlying the cochlear duct and in the cells lining the nerve pathway. EphB1 and its potential ligands ephrin-B1 and ephrin-B2 showed a segregated layered expression in the lateral wall of the cochlear duct (the external sulcus), which together with EphA4 expressed in the area, form a four-layered structure with an alternating pattern of receptors and ligands in the different layers. This arrangement gives the potential for different bidirectional Eph-mediated interactions between each of the layers. The results suggest that the Eph system in the cochlea may have a role in maintaining cell segregation during phases of cochlear development.     

Characterization of leukocyte subtypes in chicken inner ear sensory epithelia

Human hearing and balance require intact inner ear sensory hair cells, which transduce mechanical stimuli into electrical signals that are transmitted to the brain. Loss of hair cells after birth in mammals is irreversible, whereas birds are able to regenerate hair cells after insult and demonstrate ongoing hair cell production in the vestibular epithelia. Leukocytes reside in undamaged sensory epithelia of the avian inner ear and increase in number after trauma, prior to the proliferation of hair cell progenitors. It has been hypothesized that leukocyte-produced growth factors or cytokines may be involved in triggering hair cell regeneration. Little is known about the specific leukocyte subtypes present in avian ear. Immunohistochemistry with a panel of monoclonal antibodies to chicken leukocytes was used to identify leukocyte subtypes in normal posthatch chicken ear sensory epithelia. The responsiveness of the leukocytes to aminoglycoside-induced damage was also observed. Based on immunocytochemical and morphological criteria, we quantified leukocyte subtypes in normal and drug-damaged auditory and vestibular sensory epithelia. Data indicate that lymphocytes (B and T cells) do not reside in normal or drug-damaged ear sensory epithelia at 1-3 days post insult but are present in adjacent nonsensory tissues. The most common leukocytes in inner ear sensory epithelia are ramified cells of the myeloid lineage. Many of these are MHC class II positive, and a small percentage are mature tissue macrophages. An absence of leukocytes in lesioned areas of the auditory sensory epithelium suggests they may not play a critical role in triggering hair cell regeneration.     

Quantitative analyses of postembryonic hair cell addition in the otolithic endorgans of the inner ear of the european hake,merluccius merluccius (gadiformes,teleostei)

Bony fishes add sensory hair cells to the saccule and lagena of the ear for at least several years after hatching. However, it is not known whether hair cell proliferation occurs for the whole lifetime of an animal, whether proliferation occurs in all endorgans of the ear, or whether the rate of proliferation is the same in all of the endorgans. To obtain answers to these questions, the extent of postembryonic hair-cell proliferation was determined in the saccule, lagena, and utricle of the ear in the European hake, Merluccius merlaccius, for fish ranging from 7 to 75 cm in total length (6 months to 9 years of age). Results demonstrated that hair-cell addition continued throughout this period in all three otic endorgans, although endorgan size was proportionally greatest in smaller animals. Of the three endorgans, cell addition was greatest in the saccule. Moreover, far more cells were added to the caudal end of the saccule than to the rostral end. Each saccule of the largest hake had over 900,000 hair cells. It is estimated that each saccule adds approximately 110,000 new hair cells each year (or 302 cells/day) over the life span of the fish studied. A significant number of small ciliary bundles, thought to represent newly proliferated hair cells, was found throughout each endorgan, and the number of such bundles declined as the rate of hair cell proliferation decreased. The results demonstrate that extensive proliferation occurs in all three otolithic endorgans of the ears in a fish and that such proliferation continues for virtually the whole life of the animal. The functional significance of this addition is not known. © 1994 Wiley-Liss, Inc.     

Contribution of bone marrow hematopoietic stem cells to adult mouse inner ear: mesenchymal cells and fibrocytes

Bone marrow (BM)-derived stem cells have shown plasticity with a capacity to differentiate into a variety of specialized cells. To test the hypothesis that some cells in the inner ear are derived from BM, we transplanted either isolated whole BM cells or clonally expanded hematopoietic stem cells (HSCs) prepared from transgenic mice expressing enhanced green fluorescent protein (EGFP) into irradiated adult mice. Isolated GFP(+) BM cells were also transplanted into conditioned newborn mice derived from pregnant mice injected with busulfan (which ablates HSCs in the newborns). Quantification of GFP(+) cells was performed 3-20 months after transplant. GFP(+) cells were found in the inner ear with all transplant conditions. They were most abundant within the spiral ligament but were also found in other locations normally occupied by fibrocytes and mesenchymal cells. No GFP(+) neurons or hair cells were observed in inner ears of transplanted mice. Dual immunofluorescence assays demonstrated that most of the GFP(+) cells were negative for CD45, a macrophage and hematopoietic cell marker. A portion of the GFP(+) cells in the spiral ligament expressed immunoreactive Na, K-ATPase, or the Na-K-Cl transporter (NKCC), proteins used as markers for specialized ion transport fibrocytes. Phenotypic studies indicated that the GFP(+) cells did not arise from fusion of donor cells with endogenous cells. This study provides the first evidence for the origin of inner ear cells from BM and more specifically from HSCs. The results suggest that mesenchymal cells, including fibrocytes in the adult inner ear, may be derived continuously from HSCs.     

Development of calretinin immunoreactivity in the mouse inner ear

Calretinin is a calcium-binding protein of the EF-hand family. It has been previously identified in particular cell types of adult guinea pig, rat, and chinchilla inner ear. Development of calretinin immunoreactivity in the mouse inner ear was investigated from embryonic day 13 (E13) to the adult stage. In the adult mouse vestibule, calretinin immunoreactivity was present in the same structures as described for the rat and guinea pig: the population of afferent fibers forming calyx units and small number of ganglion neurons. The earliest immunoreactivity was found at E17 in vestibular hair cells (VHCs), then, at E19, in afferent fibers entering the sensory epithelia and in rare ganglion neurons. At postnatal day 4 (P4), a few vestibular nerve fibers and ganglion neurons were reactive. From this stage until P14, immunoreactivity developed in the calyx units and disappeared from VHCs. At P14, immunostaining was adult-like. In the adult mouse cochlea, immunoreactivity was present in the same cell populations as described in the rat: the inner hair cells (IHCs) and most of Corti's ganglion neurons. Calretinin immunoreactivity appeared at E 19-P0 in IHCs and ganglion neurons of the basal turn. At P1, outer hair cells (OHCs) of the basal turn were positive. Calretinin immunoreactivity then appeared in IHCs, OHCs, and ganglion neurons of the medial turn, then of the apical turn. At P4, all IHCs and OHCs and most of the ganglion neurons were immunostained. Immunoreactivity gradually disappeared from the OHCs starting at P10 and, at P22, only IHCs and ganglion neurons were positive. The sequences of appearance of calretini, n were specific to each cell type of the inner ear and paralleled their respective maturation. Calretinin was transiently expressed in VHCs and OHCs. © 1994 Wiley-Liss, Inc.     

Cellular distribution of the Fragile X mental retardation protein in the inner ear: a developmental and comparative study in the mouse,rat, gerbil,and chicken

The Fragile X mental retardation protein (FMRP) is an mRNA binding protein that is essential for neural circuit assembly and synaptic plasticity. Loss of functional FMRP leads to Fragile X syndrome (FXS), a neurodevelopmental disorder characterized by sensory dysfunction including abnormal auditory processing. While the central mechanisms of FMRP regulation have been studied in the brain, whether FMRP is expressed in the auditory periphery and how it develops and functions remains unknown. In this study, we characterized the spatiotemporal distribution pattern of FMRP immunoreactivity in the inner ear of mice, rats, gerbils, and chickens. Across species, FMRP was expressed in hair cells and supporting cells, with a particularly high level in immature hair cells during the prehearing period. Interestingly, the distribution of cytoplasmic FMRP displayed an age-dependent translocation in hair cells, and this feature was conserved across species. In the auditory ganglion (AG), FMRP immunoreactivity was detected in neuronal cell bodies as well as their peripheral and central processes. Distinct from hair cells, FMRP intensity in AG neurons was high both during development and after maturation. Additionally, FMRP was evident in mature glial cells surrounding AG neurons. Together, these observations demonstrate distinct developmental trajectories across cell types in the auditory periphery. Given the importance of peripheral inputs to the maturation of auditory circuits, these findings implicate involvement of FMRP in inner ear development as well as a potential contribution of periphery FMRP to the generation of auditory dysfunction in FXS.     

Developmental expression of the actin depolymerizing factor ADF in the mouse inner ear and spiral ganglia

Hair cells, the inner ear's sensory cells, are characterized by tens to hundreds of actin‐rich stereocilia that form the hair bundle apparatus necessary for mechanoelectrical transduction. Both the number and length of actin filaments are precisely regulated in stereocilia. Proper cochlear and vestibular function also depends on actin filaments in nonsensory supporting cells. The formation of actin filaments is a dynamic, treadmill‐like process in which actin‐binding proteins play crucial roles. However, little is known about the presence and function of actin binding molecules in the inner ear, which set up, and maintain, actin‐rich structures and regulate actin turnover. Here we examined the expression and subcellular location of the actin filament depolymerizing factor (ADF) in the cochlea and vestibular organs. By means of immunocytochemistry and confocal microscopy, we analyzed whole‐mount preparations and cross‐sections in fetal and postnatal mice (E15–P26). We found a transient ADF expression in immature hair cells of the organ of Corti, the utricle, and the saccule. Interestingly, the stereocilia were not labeled. By P26, ADF expression was restricted to supporting cells. In addition, we localized ADF in presynaptic terminals of medio‐olivocochlear projections after hearing onset. A small population of spiral ganglion neurons strongly expressed ADF. Based on their relative number, peripheral location within the ganglion, smaller soma size, and coexpression of neurofilament 200, we identified these cells as Type II spiral ganglion neurons. The developmentally regulated ADF expression suggests a temporally restricted function in the stereocilia and, thus, a hitherto undescribed role of ADF. J. Comp. Neurol. 518:1724–1741, 2010. © 2009 Wiley‐Liss, Inc.     

Synaptic proteins are tonotopically graded in postnatal and adult type I and type II spiral ganglion neurons

Inherent in the design of the mammalian auditory system is the precision necessary to transduce complex sounds and transmit the resulting electrical signals to higher neural centers. Unique specializations in the organ of Corti are required to make this conversion, such that mechanical and electrical properties of hair cell receptors are tailored to their specific role in signal coding. Electrophysiological and immunocytochemical characterizations have shown that this principle also applies to neurons of the spiral ganglion, as evidenced by distinctly different firing features and synaptic protein distributions of neurons that innervate high- and low-frequency regions of the cochlea. However, understanding the fine structure of how these properties are distributed along the cochlear partition and within the type I and type II classes of spiral ganglion neurons is necessary to appreciate their functional significance fully. To address this issue, we assessed the localization of the postsynaptic AMPA receptor subunits GluR2 and GluR3 and the presynaptic protein synaptophysin by using immunocytochemical labeling in both postnatal and adult tissue. We report that these presynaptic and postsynaptic proteins are distributed oppositely in relation to the tonotopic map and that they are equally distributed in each neuronal class, thus having an overall gradation from one end of the cochlea to the other. For synaptophysin, an additional layer of heterogeneity was superimposed orthogonal to the tonotopic axis. The highest anti-synaptophysin antibody levels were observed within neurons located close to the scala tympani compared with those located close to the scala vestibuli. Furthermore, we noted that the protein distribution patterns observed in postnatal preparations were largely retained in adult tissue sections, indicating that these features characterize spiral ganglion neurons in the fully developed ear.     

Structural changes in the inner ear over time studied in the experimentally deafened guinea pig

Today a cochlear implant (CI) may significantly restore auditory function, even for people with a profound hearing loss. Because the efficacy of a CI is believed to depend mainly on the remaining population of spiral ganglion neurons (SGNs), it is important to understand the timeline of the degenerative process of the auditory neurons following deafness. Guinea pigs were transtympanically deafened with neomycin, verified by recording auditory brainstem responses (ABRs), and then sacrificed at different time points. Loss of SGNs as well as changes in cell body and nuclear volume were estimated. To study the effect of delayed treatment, a group of animals that had been deaf for 12 weeks was implanted with a stimulus electrode mimicking a CI, after which they received a 4‐week treatment with glial cell‐derived neurotrophic factor (GDNF). The electrical responsiveness of the SGNs was measured by recording electrically evoked ABRs. There was a rapid degeneration during the first 7 weeks, shown as a significant reduction of the SGN population. The degenerative process then slowed, and there was no difference in the amount of remaining neurons between weeks 7 and 18. © 2016 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc.     

Postnatal development of NT3 and TrkC in mouse ventral cochlear nucleus

In the developing nervous system, neurotrophin 3 (NT3) and brain‐derived neurotrophic factor (BDNF) have been shown to interact with each other and with different parts of a neuron or glia and over considerable distances in time and space. The auditory system provides a useful model for analyzing these events, insofar as it is subdivided into well‐defined groups of specific neuronal types that are readily related to each other at each stage of development. Previous work in our laboratory suggested that NT3 and its receptor TrkC in the mouse cochlear nucleus (CN) may be involved in directing neuronal migration and initial targeting of inputs from cochlear nerve axons in the embryo. NT3 is hard to detect soon after birth, but TrkC lingers longer. Here we found NT3 and TrkC around P8 and the peak around P30. Prominent in ventral CN, associated with globular bushy cells and stellate cells, they were localized to different subcellular sites. The TrkC immunostain was cytoplasmic, and that of NT3 was axonal and perisomatic. TrkC may be made by CN neurons, whereas NT3 has a cochlear origin. The temporal pattern of their development and the likelihood of activity‐dependent release of NT3 from cochlear axons suggest that it may not be critical in early synaptogenesis; it may provide long‐term trophic effects, including stabilization of synapses once established. Activity‐related regulation could coordinate the supply of NT3 with inner ear activity. This may require interaction with other neurotrophins, such as BDNF. © 2009 Wiley‐Liss, Inc.     

Developmental differentiation of MAP2 expression in the central versus the peripheral and efferent projections of the inner ear.

The goal of this study was to extend our knowledge of MAP2 localization in the peripheral nervous system of mammals, since most results on MAP2 distribution are obtained in the central nervous system (CNS). This study shows the presence of microtubule-associated protein 2b (MAP2b) and MAP2c in the inner ear and describes the immunocytochemical distribution of MAP in adult and developing spiral ganglion of the rat by using a well-characterized antibody for MAP2a and MAP2b. (This antibody does not recognize the immature MAP2c). MAP2 labeling is already present in spiral ganglion neurons at 16 days of gestation. From this stage and up to the first postnatal week, MAP2 labeling was strong in all spiral ganglion neurons and their central processes. Double immunostaining at the 16-day stage with anti-MAP2 and anti-neurofilament (NF) antibodies mainly showed NF labeling in central branches that corresponded to anatomically and functionally described axons of spiral neurons. The peripheral branches lacked MAP2 labeling. In neonatal and postnatal stages, MAP2 reactivity was located in spiral ganglion perikarya and their neurites. The intensity of adult labeling was, however, lower than in younger animals. The antibody used in this study did not label axons originating in the CNS as seen by a negative response in efferent fibers from the intraganglionic spiral bundle of the cochlea. Our results suggest that during ontogenesis, MAP2 is highly expressed in the central projection of spiral ganglion neurons, and then is reduced to lower quantities in the central branch after the first postnatal week and persists into adulthood.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expression of brain-derived neurotrophic factor and neurotrophin 3 mRNA in lingual papillae and taste buds indicates roles in gustatory and somatosensory innervation

Although many studies have demonstrated the dependency of taste bud function and/or survival on intact innervation, relatively few have dealt with the development of taste bud innervation. Using in situ hybridization histochemistry, we show that brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) mRNA are expressed in a specific pattern in the taste buds, tongue papillae, and lingual epithelium during development and that expression persists into adulthood. BDNF mRNA is expressed in a fraction of the taste cells of the developing and adult taste buds in rats, showing different labeling intensities among the labeled cells. NT3 mRNA seems to be located in areas other than those where BDNF mRNA is expressed, mainly in the superior epithelial surfaces of circumvallate papillae, the outer surface epithelium of foliate papilae, the superior surface and the lateral epithelium of the fungiform papillae, and the epithelium of the filiform papillae. NT3 mRNA labeling is also observed among muscle and connective tissue of the tongue. The morphological appearance, expression of NT3 mRNA, and ramification of nerve fibers in defined epithelial structures in the posterior wall of the anterior filiform papillae suggest the existence of a mechanosensory apparatus in these papillae. Nerve growth factor and neurotrophin 4 probes did not give rise to selective labeling in tongue, although their presence cannot be totally excluded. Based on present and prior studies, we suggest that BDNF is needed during initiation and for maintenance of gustatory innervation of taste buds and gustatory papillae and that NT3 is mainly needed for somatosensory innervation of the tongue. © 1996 Wiley-Liss, Inc.     

Calretinin and calbindin distribution patterns specify subpopulations of type I and type II spiral ganglion neurons in postnatal murine cochlea

As the first neural element in the auditory pathway, neurons in the spiral ganglion shape the initial coding of sound stimuli for subsequent processing. Within the ganglion, type I and type II neurons form divergent and convergent innervation patterns, respectively, with their hair cell sensory receptors, indicating that very different information is gathered and conveyed. Layered onto these basic innervation patterns are structural and electrophysiological features that provide additional levels of processing multifaceted sound stimuli. To understand the nature of this additional complexity of signal coding, we characterized the distribution of calretinin and calbindin, two regulators of intracellular calcium that serve as markers for neuronal subpopulations. We showed in acute preparations and in vitro that calretinin and calbindin staining levels were heterogeneous. Immunocytochemical analysis of colocalization further showed that high levels of staining for the two molecules rarely overlapped. Although varied amounts of calbindin and calretinin were found within each tonotopic location and neuronal type, some distinct subdistributions were noted. For example, calretinin levels were highest in neurons innervating the midcochlea region, whereas calbindin levels were similar across the entire ganglion. Furthermore, we noted that apical type II neurons, identified by antiperipherin labeling, had significantly lower levels of calretinin and higher levels of calbindin. We also established that the endogenous firing feature of onset tau of the subthreshold response showed a pattern related to quantified calretinin and calbindin staining levels. Taken together, our results suggest an additional dimension of complexity within the spiral ganglion beyond that currently categorized. J. Comp. Neurol. 522:2299–2318, 2014. © 2014 Wiley Periodicals, Inc.     

Pre- and postnatal expression of glycoconjugates (3-fucosyl-N-acetyllactosamine and HNK-1 epitopes) in the mouse inner ear

The distribution of two glycoconjugates 3-fucosyl-N-acetyllactosamine (CD15) and HNK-1 epitope (CD57) in the inner ear of the NMRI mouse was analysed from the eighth day of gestation to the 16th day after birth. CD15 epitope distribution is developmentally regulated. The up- and down-regulation of expression, the change in the number of cells which are positive, the ingrowth of CD15-positive cells and their distribution, intracellular and/or cell-surface-associated expression, all assume a characteristic appearance at each developmental stage. Distribution of CD57 documented the nerve outgrowth and formation of the innervation of the vestibular apparatus and cochlear duct. Correlation between CD15 and CD57 expression patterns revealed differences in the interaction of the ingrowing fibres and epithelial tissue between the vestibular organ and the cochlea and differences in the development of the cristae and maculae.