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Oxidative stress plays an important role in several inner ear diseases and normal aging. Nuclear (erythroid-derived 2)-like 2, also known as Nrf2, is a transcription factor encoded by the NFE2L2 gene that controls the expression of genes necessary to reduce oxidative stress. There are no studies to the date on the localization of Nrf2 in the human inner ear in normal or pathological conditions. Therefore, we investigated the immunohistochemical localization of Nrf2 in the human cochlea and vestibule using formalin-fixed celloidin-embedded human temporal bone sections. Nrf2 immunoreactivity (IR) was found in the inner and outer hair cells and supporting cells of the organ of Corti throughout the cochlea. Nfr2-IR was also found in hair cells and supporting cells of the maculae and cristae vestibular sensory epithelia. Nrf2-IR was decreased in the organ of Corti of older age individuals. The immunolocalization of Nrf2 in both auditory and vestibular sensory epithelia suggest that this transcription factor may play a relevant role in protecting sensory hair cells from oxidative stress., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Neuroglobin (Ngb) is an oxygen-binding protein with a demonstrated role in endogenous neuroprotective mechanisms. It has been shown to function as a scavenger for reactive oxidizing species thereby assisting in cellular defense against oxidative stress. In the present study, we characterized the presence of Ngb in the human cochlea. Immunohistochemical staining was performed on formalin fixed celloidin human cochlea sections obtained from human temporal bones, using affinity purified polyclonal antibodies against Ngb. Thirty-six temporal bones were analyzed, 15 with normal otologic histories and 21 diagnosed with different inner ear pathologies. Ngb immunoreactivity (Ngb-IR) was consistently expressed in the neurons of spiral ganglia (SG) and supporting cells of the organ of Corti. There was a significant decrease of Ngb-IR in SGNs from specimens with inner ear pathologies when compared to normal specimens. In contrast, Ngb-IR in the organ of Corti did not show significant changes between pathological and normal specimens. The differential pattern of Ngb expression in these cochlear structures suggests that Ngb may participate in defense mechanisms in inner ear pathologies where oxidative stress is involved., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Planar cell polarity (PCP) signaling regulates cochlear extension and coordinates orientation of sensory hair cells in the inner ear. Retroviral-mediated introduction of the Math1 transcription factor leads to the transdifferentiation of some mature supporting cells into hair cells. Testosterone, a gonadal sex steroid hormone, is associated with neuroprotection and regeneration in Central Nervous System (CNS) development. Experiments were performed in vitro using Ad5-EGFP-Math1/Ad5-Math1 in neonatal mouse cochleas. Establishment of ectopic hair-cell like cell(HCLC) polarity in the lesser epithelial ridge (LER) with or without testosterone-3-(O-carboxymethyl) oxime bovine serum albumin (testosterone-BSA) treatment was investigated to determine the role of the PCP pathway in regulating ectopic regenerated (HCLCs) through induction by Math1 and testosterone treatment. After Math1 infection, new ectopic regenerated HCLCs were detected in the LER. After the HCLCs developed actin-rich stereocilia, the basal bodies moved from the center to the distal side. Moreover, the narrower, non-sensory LER region meant that the convergent extension (CE) was also established after transfection with Math1. After 9 days of in vitro testosterone-BSA treatment, more Edu(+), Sox2(+), and HCLC cells were observed in the LER with an accompanying downregulation of E-cadherin. Interestingly, the CE of the Ad5-EGFP-math1 treated LER is altered, but the intrinsic cellular polarity of the HCLCs is not obviously changed. In summary, our results indicate that PCP signaling is involved in the development of ectopic HCLCs and the CE of the ectopic sensory region is altered by testosterone-BSA through downregulation of cell-cell adhesion. Testosterone-BSA and Math1 treatment could promote an increase in HCLCs in the LER through proliferation and transdifferentiation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

This study tested the hypothesis that hydrophilic chemotypes of the medicinal vine Uncaria tomentosa (UT) would facilitate recovery of sensorineural functions following exposure to a damaging level of noise. The particular chemotypes investigated were carboxy alkyl esters (CAE) which are known to exhibit multifunctional cytoprotective properties that include: enhanced cellular DNA repair, antioxidation and anti-inflammation. Long-Evans rats were divided into four treatment groups: vehicle-control, noise-only, CAE-only and CAE+noise. The noise exposure was an 8kHz octave band of noise at 105dB SPL for 4h. Outer hair cell (OHC) function was measured with the cubic 2f(1)-f(2) distortion product otoacoustic emissions (DPOAE) at the start of the study (baseline) and at time-points that corresponded to 1day, 1week and 4weeks post-noise exposure to determine within-group effects. Compound action potentials to puretone stimuli were recorded from the VIIIth craniofacial nerve at 4weeks post-noise exposure to determine between-group effects. Additionally, cytocochleograms were constructed for each row of OHCs from each group. Noise exposure produced significant sensorineural impairments. However, CAE treatment facilitated almost complete recovery of OHC function and limited the magnitude of cell loss. The loss of neural sensitivity to puretone stimuli was inhibited with CAE treatment. Therefore, it appears that the multifunctional cytoprotective capacity of CAE from UT may generalize to otoprotection from acoustic over-exposure., (Published by Elsevier B.V.)

The distribution of neuroglobin (Ngb) was investigated in the normal rat cochlea using immunohistochemistry and non-radioactive insitu hybridization. We also determined whether chronic, very mild CO exposure at 25ppm in air over the gestational and postnatal period alters the expression of Ngb. Pregnant rats were exposed chronically to CO from gestational days 5-20. Four groups were made as follows: prenatal exposure to CO only; prenatal exposure to CO followed by postnatal exposure from postnatal days (5) P5 to P20; rat pups were exposed to CO from P5 to P20; controls (air without CO). In normal adult rats and control group pups, Ngb was found in spiral ganglia neurons, fibrocytes of the spiral ligament, and supporting cells of the organ of Corti. Ngb was not present in the stria vascularis and the inner and outer hair cells. At P20 Ngb immunoreactivity and transcript expression decreased in spiral ganglia neurons and the spiral ligament in the prenatal and pre- and postnatal groups. This decrease was not observed in the postnatal group. Ngb-IR did not decrease in supporting cells in any CO group. Cytochrome-C immunoreactivity followed Ngb distribution in normal controls and CO treated groups. A decrease in Ngb in spiral ganglia neurons and rat spiral ligament, but not in supporting cells, following CO exposure supports the idea that chronic, mild exposure to CO may create a vulnerable cellular environment predisposed to adverse cochlear development.

Membrane leakage has been found in hair cells undergoing apoptosis following exposure to intense noise. However, it is not known whether this membrane damage is the consequence of apoptotic degeneration or direct mechanical stress. The current study was designed to investigate whether membrane damage occurred before the onset of apoptosis and to determine the level of the membrane damage. Chinchillas were exposed to an impulse noise at 155 dB peak SPL. The noise-induced membrane damage was assessed functionally, using membrane tracers with graded molecular sizes (propidium iodide and FITC-dextrans with molecular sizes of 3, 40, 500, and 2000 kDa), and morphologically, using DiO staining and semithin sections. The study revealed two major findings. First, exposure to intense noise caused a rapid increase in membrane permeability, and the onset of membrane leakage preceded the manifestation of nuclear condensation. This indicates that the early membrane damage observed in apoptosis is the direct consequence of mechanical stress. Second, the level of membrane damage was severe, allowing the entry of 3 kDa and 40 kDa FITC-dextrans, but the membrane was not completely broken down, as evidenced by the preservation of the ability to exclude 500 kDa and 2000 kDa FITC-dextran molecules and the maintenance of the cell boundary. Notably, despite the membrane damage, hair cells continue to undergo the apoptotic process, leading to the generation of a type of apoptosis with early membrane damage.

Tetraethylammonium (TEA)-sensitive potassium currents in the cochlear inner hair cells (IHCs) possess the kinetics of fast inactivation. IHCs of guinea-pigs were separately isolated from the apical and basal turns and the tonotopic gradient of inactivation kinetics was investigated. TEA-sensitive potassium currents showed voltage-dependent time constant of the inactivation phase both in apical and basal IHCs, however, the degree of inactivation (compared to the ratio between the steady-state current and initial peak current) was voltage-independent. Inactivation time constant was faster in basal IHCs than in apical IHCs and the degree of inactivation was greater in basal IHCs than in apical IHCs, suggesting that inactivation was more predominant in basal IHCs than in apical IHCs.

The spiral ganglion accommodates the cell bodies of the acoustic nerve fibres connecting the hair cells to the central nervous system. As the ionic channels containing various voltage-gated K+ channel (Kv) subunits play pivotal roles in determining the functional properties and firing behaviour of the spiral ganglion cells (SGCs), every piece of information concerning the Kv expression of the SGCs is valuable. In the present work a comprehensive immunohistochemical analysis was performed to describe the expression of 9 Kv subunits in the guinea pig cochlea on traditional wax-embedded sections as well as employing a newly developed preparation that allowed confocal analysis, reconstruction of the three-dimensional appearance and precise morphological characterisation of the SGCs. Besides determining their Kv expression patterns, differences between type I and type II SGCs were sought. SGCs showed positivity for 8 out of the 9 Kv subunit-specific antibodies with varying intensity and proportion of the immunopositive cells; whereas no obvious Kv3.2 positivity could be noted. Type I and type II cells demonstrated similar expression patterns for all subunits tested, with the exception of Kv1.2, whose presence was confirmed in only 50% of the type II cells. Although the present findings suggest that type I and type II cells do not differ fundamentally in the Kv subunits they possess; they also imply that SGCs may not form a homogeneous cell population, and might provide explanation of the previously noted heterogeneity of the membrane properties of the SGCs.

Orphanin FQ/nociceptin (OFQ/N) and its receptor (ORL-1) have been proposed to play a role in the regulation of hearing. In this study, we investigate the localization of OFQ/N-like immunoreactivity in the mammalian cochlea. Sprague-Dawley rat temporal bones were harvested and decalcified. The organ of Corti was microdissected, and indirect immunohistochemistry was performed using a rabbit polyclonal antibody raised against OFQ/N. Immunoreactivity was seen in the tunnel crossing fibers and the large boutons terminating onto outer hair cells, and in the fibers terminating onto the afferents to the inner hair cells. The findings are consistent with OFQ/N expression in lateral and medial olivocochlear efferents.

The perceptions of sound, balance and acceleration are mediated through the vibration of stereociliary bundles located on the lumenal surfaces of mechanosensory hair cells located within the inner ear. In mammals, virtually all hair cells are generated during a relatively brief period in embryogenesis with any subsequent hair cell loss leading to a progressive and permanent loss of sensitivity. In light of the importance of these cells, considerable effort has been focused on understanding the molecular genetic pathways that regulate their development. The results of these studies have begun to elucidate the signaling molecules that regulate several key events in hair cell development. In particular, significant progress has been made in the understanding of hair cell commitment, survival and differentiation. In addition, several aspects of the development of the stereociliary bundle, including its elongation and orientation, have recently been examined. This review will summarize results from each of these developmental events and describe the molecular signaling pathways involved.

Expression of the cyclin-dependent kinase inhibitor p27(Kip1) defines a post-mitotic population of cells in the embryonic mammalian cochlea that constitutes the nascent organ of Corti. Here, we describe techniques to purify these precursors using a transgenic p27/GFP reporter and fluorescence activated cell sorting (FACS). We demonstrate that these cells express other markers of the sensory lineage, such as Sox2, and when placed in dissociated cell culture differentiate as hair cells and supporting cells. The purified sensory progenitors thus obtained provide a means of studying the process of hair cell and supporting cell differentiation in vitro, as well as providing a means of analyzing the molecular and physiological properties of this unique population of cells.

Prenatal cocaine exposure causes alterations in auditory brainstem response in children and experimental animals and has adverse effects on auditory information processing and language skills in children. These effects may result from lesions in the cochlea since this organ is particularly sensitive to chemical insults during the development. We have thus studied here the effect of prenatal cocaine exposure on the maturation of the rat cochlea using the transient non-catecholaminergic expression of tyrosine hydroxylase in spiral ganglion neurons as an index of cochlear maturation and morphometry to evaluate the maturation of primary auditory neurons and the organ of Corti. We showed that prenatal cocaine exposure accelerated the cochlear maturation. In the basal coil of cochleas from PND8 cocaine-treated pups, the Kölliker's organ had disappeared, the tunnel of Corti was opened, and the stria vascularis no longer contained undifferentiated marginal cells. The maximum expression of tyrosine hydroxylase in type I primary auditory neurons occurred at PND8 instead of PND12 in pair-fed controls. On the other hand, the prenatal cocaine exposure had no effect on the width and height of the organ of Corti, spiral ganglion volume and number and size of primary auditory neurons. In conclusion, our data suggest that prenatal cocaine exposure, though not lethal to primary auditory neurons, accelerates aspects of the cochlear sensorineural maturation. This accelerated cochlear maturation in cocaine-treated rat pups could cause auditory dysfunctions by desynchronizing the development of the whole auditory pathway.

Recently, a negative feedback effect of nitric oxide (NO) on the adenosine 5'-triphosphate (ATP)-induced Ca2+ response has been described in cochlear inner hair cells. We here investigated the role of NO on the ATP-induced Ca2+ response in outer hair cells (OHCs) of the guinea pig cochlea using the NO-sensitive dye DAF-2 and Ca2+ -sensitive dye fura-2. Extracellular ATP induced NO production in OHCs, which was inhibited by L-NG-nitroarginine methyl ester (L-NAME), a non-specific NO synthase (NOS) inhibitor, and suramin, a P2 receptor antagonist. ATP failed to induce NO production in the Ca2+ -free solution. S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, enhanced the ATP-induced increase of the intracellular Ca2+ concentrations ([Ca2+]i), while L-NAME inhibited it. SNAP accelerated ATP-induced Mn2+ quenching in fura-2 fluorescence, while L-NAME suppressed it. 8-Bromoguanosine-cGMP, a membrane permeable analog of cGMP, mimicked the effects of SNAP. 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one, an inhibitor of guanylate cyclase and KT5823, an inhibitor of cGMP-dependent protein kinase inhibited the ATP-induced [Ca2+]i increase. Selective neuronal NOS inhibitors, namely either 7-nitro-indazole or 1-(2-trifluoromethylphenyl) imidazole, mimicked the effects of L-NAME regarding both ATP-induced Ca2+ response and NO production. Immunofluorescent staining of neuronal nitric oxide synthase (nNOS) in isolated OHCs showed the localization of nNOS in the apical region of OHCs. These results suggest that the ATP-induced Ca2+ influx via a direct action of P2X receptors may be the principal source for nNOS activity in the apical region of OHCs. Thereafter, NO can be produced while conversely enhancing the Ca2+ influx via the NO-cGMP-PKG pathway by a feedback mechanism.

Mouse embryonic stem (ES) cells were transplanted into the cochlea of adult guinea pigs in order to explore their survival, differentiation, and possible integration with the host tissue. With the purpose of investigating the possible effect of manipulating the local embryonic microenvironment, ES cells were transplanted into the cochlea with or without an embryonic neuronal cograft consisting of dorsal root ganglion (DRG) tissue. To detect the survival and differentiation of ES cells, cells expressing green fluorescent protein (GFP) were used in combination with immunohistochemical detection of a neuronal marker, neural class III beta-tubulin (TUJ1 antibody). At 4 weeks following transplantation implanted ES cells were found close both to the sensory epithelium, and the spiral ganglion neurons (SGNs) with their peripheral dendritic processes projecting to the organ of Corti. There was a significant difference in the number of surviving TUJ1 (+) ES cells between the DRG cograft group and the non-cograft group (P < 0.01, ANOVA). Neurite-like projections were also identified between TUJ1-positive ES cells and the peripheral dendritic processes from SGNs. The results suggest that an embryonic neuronal microenvironment may be one of the key factors in the survival and differentiation of ES cells in the adult auditory system.

Afferent synapses on inner hair cells (IHC) transfer auditory information to the central nervous system (CNS). Despite the importance of these synapses for normal hearing, their response to cochlear disease and dysfunction is not well understood. The C57BL/6J mouse is a model for presbycusis and noise-induced hearing loss because of its age-related hearing loss and susceptibility to acoustic over-exposure. In this context, we sought to establish normal synaptic structure in order to better evaluate synaptic changes due to presbycusis and noise exposure. Ultrastructural analysis of IHCs and afferent terminals was performed in a normal hearing 3-month-old C57BL/6J mouse at cochlear sites corresponding to 8, 16 and 32 kHz using semi-serial sections. A stereologic survey of random sections was conducted of IHCs in 11 additional mice. Two morphologically distinct groups of afferent terminals were identified at all 3 frequency locations in 11 out of 12 animals. "Simple" endings demonstrated classic features of bouton terminals, whereas "folded" endings were larger in size and exhibited a novel morphologic feature that consisted of a fully internalized double membrane that partially divided the terminal into two compartments. In many cases, the double membrane was continuous with the outer terminal membrane as if produced by an invagination. We still must determine the generality of these observations with respect to other mouse strains.

Traditional methods for anatomical and morphometric studies of cochlear tissues have relied upon either microdissection of the organ of Corti or the generation of serial sections of the cochlea. Such methods are time-consuming, disruptive to three-dimensional relationships and often restrict sampling to very limited numbers of cells. We have found that cells and tissue components of the cochlear duct may be labelled by fluorescent markers within intact cochleae, which are then embedded in epoxy resin for subsequent viewing by fluorescent microscopy methods. This approach allows imaging through thick optical volumes with preservation of three-dimensional relationships. Unlike sectioned tissue, alignment of the sample relative to the focal axis may be easily corrected by re-orientation of the optical volume with common image processing software. Fluorescently labelled cochleae embedded in epoxy can be viewed by most fluorescent microscopy methods including laser scanning confocal microscopy, multi-photon confocal microscopy and widefield epi-fluorescence microscopy with deconvolution. Furthermore, semi-thin sections made from these preparations are compatible with traditional histological stains, as well as allowing brightly labelled epi-fluorescent images.

The efferent innervation of the auditory basilar papilla of birds and mammals is provided by a dedicated population of brainstem neurones that are separate from those supplying the vestibular organs. This study addresses the question whether a population of bilaterally-projecting efferents, contacting hair cells in both basilar papillae, is consistently present in birds. The chicken and the barn owl were chosen, two species where the total number of efferents was already known and which represent two extremes of an auditory generalist and an auditory specialist, respectively. Fluorogold and Choleratoxin, two potent retrograde tracers, were injected into one cochlear duct each of all individuals. Labelled neurones were subsequently identified in the brainstem using standard fluorescence techniques. A small proportion (up to 2% of the total population) of double-labelled cells was found in both species. The great majority of those double-labelled neurones could be assigned to the ventrolateral group of efferents, which has previously been shown to project exclusively to the auditory basilar papilla. Thus, in birds, like in mammals, a small subgroup of auditory efferents innervates both basilar papillae.

We investigated the direct action of glutamate (Glu) on the membrane current of isolated inner hair cells of guinea-pig cochlea. Glu elicited inward currents at a holding potential of -70 mV. Eight of 13 cells showed a steady inward current, while five of 13 cells showed a fast and rapidly desensitized current. I-V relationships demonstrated that the reversal potential of Glu-induced current was near 0 mV. Glu-induced currents were dose-dependent, where the half maximum concentration (K(d)) was 41 microM and Hill coefficient (n) was 1.75.

Mutations in the GJB6 gene encoding connexin 30 (Cx30) can cause dominant forms of nonsyndromic deafness. By studying immunohistochemical localization of Cx30 in the mouse cochlea at different ages from 0 to 30 days after birth, we found that the expression of Cx30 is nearly the same as that of Cx26. These findings suggest that as well as Cx26, Cx30 may also contribute to the generation and maturation of endocochlear potential.

Expression of multidrug resistance protein 1 (MRP1) was detected in the rat cochlea by RT-PCR and immunohistochemistry using anti-MRP monoclonal antibody MRPr1. Use of primers specific for rat mrp1 gene resulted in the amplification of an expected 394 bp fragment prepared from brain and cochlear tissues. Immunohistochemically, MRP was found in the choroid plexus, stria vascularis, spiral ligament, spiral prominence and cochlear nerve in the modiolus. From these results, it was suggested that MRP in the rat cochlea might function as an extrusion pump and play an important role in the blood-inner ear barrier.

Hair cells of the vertebrate inner ear are subject to efferent control by the release of acetylcholine (ACh) from brainstem neurons. While ACh ultimately causes the hair cell to hyperpolarize through the activation of small conductance Ca(2+)-activated K(+) channels, the initial effect is to open a ligand-gated cation channel that briefly depolarizes the hair cell. The hair cell's ligand-gated cation channel has unusual pharmacology that is well matched to that of the nicotinic subunit alpha9 expressed in Xenopus oocytes. We used sequence-specific amplification to identify the ortholog of alpha9 in the chick's cochlea (basilar papilla). Chick alpha9 is 73% identical to rat alpha9 at the amino acid level. A second transcript was identified that differed by the loss of 132 base pairs coding for 44 amino acids near the putative ligand-binding site. RT-PCR on whole cochlear ducts suggested that this short variant is less abundant than the full length alpha9 mRNA. In situ hybridization revealed alpha9 mRNA in sensory hair cells of the chick cochlea. The pattern of expression was consistent with the efferent innervation pattern. The alpha9 label was strongest in short (outer) hair cells on which large calyciform efferent endings are found. Tall (inner) hair cells receiving little or no efferent innervation had substantially less label. The cochlear ganglion neurons were not labeled, consistent with the absence of axo-dendritic efferent innervation in birds. These findings suggest that alpha9 contributes to the ACh receptor of avian hair cells and supports the generality of this hypothesis among all vertebrates.

Synaptophysin immunoreactivity was used as a marker for the olivocochlear efferent system that innervates the outer hair cells of the cochlea. An intense noise exposure at either 6.3 kHz or 1.0 kHz caused a significant reduction in anti-synaptophysin immunoreactivity within the 8-6 mm or 14-11 mm distance from the round window, respectively. In the region of the main lesion, the reduction in synaptophysin immunoreactivity for both the 6.3 and 1.0 kHz exposures correlated well with outer hair cell loss. In regions peripheral to the main lesion, some remnants of efferent nerve endings could remain even when their associated outer hair cells were missing. Pre-treatment with a low level sound conditioner (either at 6.3 tone or 1.0 kHz) effectively reduced the efferent and outer hair cell pathology induced by the 6.3 and 1.0 kHz intense noise exposures, respectively. The results demonstrate the feasibility of using anti-synaptophysin immunoreactivity as an effective means of quantifying pathological alterations to the medial cochlear efferent terminals throughout the cochlea. Furthermore, the results show that sound conditioning significantly reduces damage to the efferent terminals.

The distribution and density of noradrenergic (NA) and serotonergic (5-HT) varicosities in the superior olive (SO) and periolivary region (PO) and their relationship to olivocochlear neurons was studied. Antibodies against 5-HT and the NA precursor enzyme dopamine beta-hydroxylase were utilized to examine the density of innervation of SO and PO. To determine the relationship of these varicosities to efferent neurons projecting to the cochlea, olivocochlear neurons were retrogradely labeled with biotinylated dextranamine (BDA). NA and 5-HT varicosities were found adjacent to labeled olivocochlear neuron cell bodies and dendrites. More than 50% of labeled medial olivocochlear (MOC) neurons showed likely contact with NA varicosities and more than 90% of labeled MOC neurons with 5-HT varicosities. There was no apparent difference in the number of lateral olivocochlear (LOC) neurons in close proximity to NA and 5-HT varicosities versus MOCs in close proximity to NA and 5-HT varicosities. Our results suggest that the NA and 5-HT systems are in a position to modulate auditory brainstem processing. The specific relationship of NA and 5-HT varicosities to olivocochlear neurons suggests that one possible level of modulation is prior to signal transduction., (Copyright 1999 Elsevier Science B.V.)

Recent studies have shown that an attempt at auditory hair cell neodifferentiation occurs in vivo in the rat organ of Corti after amikacin intoxication during the last stages of cochlear maturation. Atypical cells, with morphological characteristics reminiscent of very immature sensory hair cells, were transiently observed after outer hair cell losses. The aim of the present study was to assess (i) if this attempt at hair cell neodifferentiation was related to the degree of maturity of the organ of Corti and (ii) to characterise morphological and molecular changes in the scarring epithelium. We therefore investigated, using electron and confocal microscopy, morphological and molecular changes in cochleae from rats treated with amikacin at two different periods: from post natal day (PND) 1 to PND 8, when the organ of Corti is very immature; and from PND 30 to 37, when the organ of Corti is morphologically and functionally mature. In both groups, transient atypical cells were observed, attesting that the attempt at hair cell neodifferentiation is not strictly related to the immaturity of the cochlea. The results also suggest that Deiters cells are involved in the appearance of atypical cells, possibly through a transdifferentiation process. Finally, it appears that non-sensory epithelial cells from the outer spiral sulcus progressively colonize the region of pre-existing outer hair cells., (Copyright 1998 Elsevier Science B.V.)

Elimination of auditory nerve activity results in atrophy and death of nucleus magnocellularis (NM) neurons in the chick. One early event in the degeneration of NM neurons is a disruption of their ribosomes. This experiment examines the role of metabotropic glutamate receptors in afferent regulation of ribosomes. The auditory nerve on one side of a chick brainstem slice was stimulated in vitro. Rapid stimulation-dependent changes in ribosomes were visualized by immunolabeling using an antibody, called Y10B, that recognizes ribosomal RNA. In normal media, NM neurons on the stimulated side of the slice show greater Y10B labeling than the unstimulated NM neurons on the opposite side of the same slice. The role of metabotropic glutamate receptors was evaluated by unilaterally stimulating the auditory nerve in media containing the metabotropic glutamate receptor antagonist (RS)-alpha-methyl-4-carboxyphenyl-glycine (MCPG). Addition of MCPG to the bath did not block EPSPs produced by stimulating the auditory nerve. However, MCPG did prevent the stimulation-dependent regulation of ribosomes in NM neurons (as indexed by Y10B labeling). These data suggest that glutamate may play a trophic role in the young auditory system through activation of metabotropic glutamate receptors.

Nitric oxide synthase (NOS) activity and NADPH diaphorase staining has previously been reported in mammalian cochlea. Here we demonstrate immunoreactivity for neuronal-type NOS I and endothelial-type NOS III in the cochlea of the guinea pig. NOS I immunoreactivity was seen in inner and outer hair cells, and spiral ganglion cells. Staining for NOS I was also shown in basal and intermediate cells of the stria vascularis, spiral ligament cells, and the media of vessels near the modiolus. An antibody to NOS III stained primarily vascular endothelial cells. Some NOS III immunoreactivity was also detected in spiral ganglion cells. An antibody to the inducible-type NOS II did not stain any structure of the guinea pig cochlea, suggesting that this isoform is not expressed under physiological conditions. Nitric oxide produced by NOS I and/or NOS III may act as a neuromodulator in the organ of Corti and could also play a role as a regulator of cochlear blood flow.

Programmed cell death (apoptosis) in the inner ear of senescence-accelerated mouse was identified using specific labeling of fragmented DNA (the TUNEL method). In spite of some inter-individual differences, the apoptotic cells were predominantly found in the phylogenetically newer part of the inner ear, the cochlea and the saccules. In the saccules, sensory hair cells as well as supporting cells were positively labeled. In the cochlea, positive staining was detected in inner and outer hair cells, pillar cells, Deiters' cells, interdental cells, the stria vascularis (marginal cells, intermediate cells, basal cells), and cells in Reissner's membrane. The present results suggest that age-related cell death, which may cause hearing impairment and dysequilibrium, is due to apoptosis occurring in the inner ear.

Electronmicroscopic immunogold cytochemistry was applied to investigate the localization of glutathione (GSH) in the guinea pig inner ear. GSH immunoreactivity was preferentially distributed in basal cells and intermediate cells of the stria vascularis, and in fibrocytes and capillary endothelial cells in the spiral ligament and vestibular endorgans. The present findings suggest that the synthesis of GSH, a peptide known to protect against ototoxic compounds, depends on restricted cell populations in the inner ear.

The development of Ca-ATPase immunoreactivity in gerbil outer hair cells (OHCs), assayed by immunofluorescence and postembedding immunocytochemistry, is reported here. In the adult, a linear array of label is seen inside the lateral plasma membrane. The ultrastructural distribution of Ca-ATPase near the OHC lateral plasma membrane was examined using immunogold cytochemistry and showed this calcium pumping enzyme to be present throughout the subsurface cisternal complex (SSC), especially near the innermost layers. During development, Ca-ATPase immunoreactivity appeared in patches near the lateral plasma membrane of some OHCs of the third row by 12 days after birth (DAB). By 15-16 DAB, punctate immunoreactivity was detected in the second and first rows. At 20 DAB, immunostaining near OHC lateral plasma membrane was increased, but was less continuous than OHC staining in the adult cochlea. The appearance of Ca-ATPase in OHCs coincides with the onset of auditory function and isolated OHC motility in the gerbil. The ultrastructural demonstration of abundant sites of calcium pumps in the SSC supports a role for this structure in the intracellular storage of calcium. These findings suggest a possible role of Ca-ATPase and the SSC in the regulation of slow motility of OHCs which has been reported to depend on intracellular calcium concentration.

Double-label techniques were used to concomitantly label olivocochlear neurons and serotoninergic fibers in the bush baby (Otolemur garnettii) brainstem. Light-microscopic examination (using a 100 x plan apochromatic oil-immersion objective) of the sections revealed that serotonin-positive varicosities (presumptive terminal endings) contacted somata and dendrites of neurons belonging to both the lateral and medial olivocochlear neurons near the superior olivary nuclei. These results provide direct evidence that the olivocochlear system (a specific auditory brainstem pathway) receives input from the serotoninergic system (a diffuse reticular brainstem network).

The age-related change in glial fibrillary acidic protein (GFAP) immunoreactivity was analyzed in young (3 months) and old (24 months) adult rat cochlear nuclei (CN). Quantitative analyses show a significant increase with age, in the number of GFAP positive astrocytes and processes in the old adult when compared with the young adult rat. There was also a differential distribution of GFAP immunoreactivity in the young adult CN where it predominates in the granular cell region, whereas in old rats, the GFAP immunoreactivity distribution was homogeneous in all parts of the nucleus. There was no change in the total number of neurons between these two stages in any part of the nucleus except for the antero-ventral CN, where a decrease in neuronal number was observed in the aged rats. The increase in GFAP immunoreactivity was related to an increase of both GFAP positive astrocyte number and processes. The increase of GFAP positive astrocytes may be due either to an alteration of auditory nerve fibers, changing the trophic interactions with post-synaptic cells, or to intrinsic alterations of CN neurons and local circuits reflecting aging of the CN.

The effects of caffeine on the outer hair cells (OHCs) freshly dissociated from guinea-pig cochlea were investigated with the whole-cell patch-clamp technique, in both the conventional and the nystatin perforated patch-clamp configurations under voltage-clamp condition. Application of caffeine (> 1 mM for 10-30 s) induced an inward current (Icaffeine) with decrease of conductance in a dose-dependent manner at a holding potential (VH) of -60 mV. The reversal potential of Icaffeine (Ecaffeine) was close to the K+ equilibrium potential. The Icaffeine was not affected by Ca(2+)-free external solution. The internal perfusion of the Ca2+ chelator BAPTA had no effect on Icaffeine. The Icaffeine was not modulated by the external application of H-8 or staurosporine and by the internal perfusion of GDP-beta S. The amplitude of Icaffeine was the largest at the basal region of OHCs when caffeine was locally applied by the 'puffer' method. These results suggest that caffeine induces a decrease in membrane potassium conductance of the OHCs mainly at the basal region without mediating the intracellular signaling pathway.

The effect of the ototoxic molecule cisplatin (cis-DDP) on the voltage-dependent Ca2+ channel in dissociated outer hair cells (OHCs) of guinea-pig cochlea was investigated using a whole-cell patch-clamp technique. Cis-DDP had antagonistic effect on the Ca2+ channel and reversibly suppressed the Ca2+ current in a concentration-dependent manner. These results suggested that one of the ototoxic mechanisms of cis-DDP is involved in the inhibition of the Ca2+ channel in OHCs.

The acute effects of the aminoglycoside antibiotics gentamicin on isolated cochlear outer hair cells (OHC) was investigated by whole-cell patch-clamp and measurements of the intracellular potassium level by means of the potassium-sensitive dye PBFI. In addition, the accompanying length changes of OHC are described. It could be shown that gentamicin at different concentrations reversibly induces a hyperpolarization by about 5-10 mV, potassium outflow from the cytoplasm (by about 22 mM) and a cellular elongation (by about 10%). It is suggested that these effects are the result of an interaction between gentamicin and the cochlear transduction channels in OHC as suggested earlier. These acute effects are distinctly different from the chronic gentamicin effects which are based on the metabolization of the antibiotics to cause the death of the OHC by interaction with the phosphoinositide signalling cascade.

The olivocochlear pathway in the developing rat was visualized in fixed material. The fluorescent carbocyanine dye 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was applied to the cut central axons of the olivocochlear neurones at the floor of the fourth ventricle, and the termination pattern within the cochlea was examined after dye diffusion. From the day of birth (P0) to postnatal day 2 (P2), efferent innervation of the cochlea was exclusively in the region of the inner hair cells. Between P2 and P11, progressive outgrowth of neuronal processes to the outer hair cell region occurred; possible connections with the outer hair cells were occasionally seen at P4 and approached the mature pattern by P6. The efferent innervation of the organ of Corti appeared to mature progressively from the cochlear base to the apex, with outgrowth to the outer hair cells occurring earlier in the basal turn of the cochlea than in the second and third cochlear turns. Numerous blind axonal endings were observed in the spiral lamina especially at early postnatal ages. These findings may be consistent with a sequential pattern of arrival of efferent axons at the organ of Corti and ongoing death of efferent neurones in the brainstem during this period of development.

In the developing inner ear, the existence of a neuronal death and of a peripheral target-derived trophic effect on cochleovestibular neurons has been documented. Using cultures of rat cochleovestibular neurons, we show that the E12 otic vesicle releases a factor promoting the survival and the neuritogenesis of these neurons, and that this effect is mimicked by NGF. The effect of the optic vesicle conditioned medium (OVCM) on cochleovestibular neurons is suppressed by anti-NGF antibodies. OVCM is neuronotrophic for NGF-sensitive sympathetic neurons, an effect that is also suppressed by anti-NGF antibodies, further demonstrating the presence of biologically active nerve growth factor.

The packing density of spiral ganglion neurons was measured in 2.5- and 13-15-month-old guinea pigs, in guinea pigs at various times after drug-deafening or acoustic trauma, and in Waltzing guinea pigs of various ages. Analysis of variance and Duncan's new multiple range tests were used to determine significant differences between treatment/survival groups. Spiral ganglion neurons in young and old normal ears did not have significantly different packing densities. Drug-deaf guinea pigs showed a significant loss of neurons by 2 weeks following treatment, a further significant loss by 2 months, and a marginally significant loss between 4 and 8 months. The neuronal population was then stable through 15 months, at about 13% of normal. Acoustic trauma ears showed the first significant loss isn the lower second turn at 1 month. Long-term (12-14 months post-exposure) trauma ears were highly variable. Waltzers lost about 50% of their normal neuronal population between 4 and 8 months; they showed an unexpected greater-than-normal density at 2 months, possible explanations of which are discussed. Thus, loss of the organ of Corti from various causes results in a slow but progressive loss of spiral ganglion neurons, the time course of which varies with the type of cochlear insult.