Loss of function of Ywhah in mice induces deafness and cochlear outer hair cells' degeneration.

In vertebrates, 14-3-3 proteins form a family of seven highly conserved isoforms with chaperone activity, which bind phosphorylated substrates mostly involved in regulatory and checkpoint pathways. 14-3-3 proteins are the most abundant protein in the brain and are abundantly found in the cerebrospinal fluid in neurodegenerative diseases, suggesting a critical role in neuron physiology and death. Here we show that 14-3-3eta-deficient mice displayed auditory impairment accompanied by cochlear hair cells' degeneration. We show that 14-3-3eta is highly expressed in the outer and inner hair cells, spiral ganglion neurons of cochlea and retinal ganglion cells. Screening of YWHAH, the gene encoding the 14-3-3eta isoform, in non-syndromic and syndromic deafness, revealed seven non-synonymous variants never reported before. Among them, two were predicted to be damaging in families with syndromic deafness. In vitro, variants of YWHAH induce mild mitochondrial fragmentation and severe susceptibility to apoptosis, in agreement with a reduced capacity of mutated 14-3-3eta to bind the pro-apoptotic Bad protein. This study demonstrates that YWHAH variants can have a substantial effect on 14-3-3eta function and that 14-3-3eta could be a critical factor in the survival of outer hair cells.

Coxsackie adenovirus receptor and alpha nu beta3/alpha nu beta5 integrins in adenovirus gene transfer of rat cochlea.

This study was designed to determine whether Coxsackie adenovirus receptor (CAR) and alpha nu beta3/alpha nu beta5 integrin co-receptors are involved in adenovirus gene transfer in the rat cochlea. We find that CAR and integrin co-receptors are expressed in every cell subtype transduced by the adenoviral vector Ad5 DeltaE1-E3/cytomegalovirus/green fluorescent protein (GFP) on cochlear slices in vitro. The spiral ganglion neurons, which do not express CAR, were not transduced by the virus. Blocking these receptors by monoclonal antibodies decreased transgene expression, whereas disrupting tight junctions with ethylenediaminetetraacetic acid led to an increased transgene expression. However, sensory hair cells and strial cells also expressing CAR and alpha nu integrins were not transduced by the vector. GFP expression was also studied in vivo. Perilymphatic perfusion of adenovirus in vivo did not affect hearing and only cells lining the perilymphatic spaces were transduced. Endolymphatic perfusion resulted in low-frequency hearing loss and although some cells of the organ of Corti were efficiently transduced, the sensory and the strial cells were not. Transduced sensory and strial cells were occasionally observed in cochleas after single shot of adenovirus. Pretreatment with anti-CAR and anti-alpha nu antibodies decreases GFP expression in vivo, suggesting that the CAR/alpha nu integrin pathway is involved in adenovirus transduction in the cochlea.

Glutamate transporters in the guinea-pig cochlea: partial mRNA sequences, cellular expression and functional implications.

In the cochlea, glutamate plays a major role in synaptic transmission between the inner hair cell and the primary auditory neurons. Extracellular glutamate concentration must be regulated to prevent excitotoxicity. This regulation is mediated by excitatory amino acid transporters, membrane proteins that remove glutamate from the synaptic cleft. In this study, we investigated the distribution and activity of three excitatory amino acid transporters subtypes in the guinea-pig cochlea: glutamate aspartate transporter, glutamate transporter and excitatory amino acid carrier. A partial messenger ribonucleic acid sequence was determined for each of these transporters, by polymerase chain reaction with degenerate primers, using guinea-pig brain complementary deoxyribonucleic acid as the template. Primers specific for each transporter were then designed and used to screen a dissected organ of Corti complementary deoxyribonucleic acid library. The cellular distribution of each transporter was examined by immunocytochemistry. We investigated the functional consequences of inhibiting glutamate uptake by recording cochlear potentials during intracochlear perfusion with either l-trans-pyrrolidine-2,4-dicarboxylic acid or dihydrokainate. At the end of the electrophysiological session, cochleas were processed for electron microscopy. Only the glutamate aspartate transporter messenger ribonucleic acid was detected in the organ of Corti. Consistently, glutamate aspartate transporter protein was detected in the inner hair cell-supporting cells and in the ganglion of Corti satellite cells. Glutamate transporter and excitatory amino acid carrier were found in the afferent auditory neurons. Only intracochlear perfusions with l-trans-pyrrolidine-2,4-dicarboxylic acid resulted in a dose-dependent decrease in the amplitude of the cochlear compound action potential, leaving cochlear microphonic potential unaffected. After l-trans-pyrrolidine-2,4-dicarboxylic acid perfusion, cochleas displayed a swelling of the afferent endings typical of excitotoxicity. [(-)1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-4,5-dihydro-3-methylcarbamyl-2,3-benzodiazepine], a selective alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist protects the cochlea against l-trans-pyrrolidine-2,4-dicarboxylic acid effect.

Expression pattern of mammalian cochlea outer hair cell (OHC) mRNA: screening of a rat OHC cDNA library.

The aim of this study was to characterize the mRNA content of mammalian cochlear outer hair cells (OHCs) and to search for specific genes possibly involved in their unique properties. Indeed, OHCs, which feature high-frequency electromotility, are responsible for the exquisite sensitivity and frequency selectivity of the cochlea. Damage to these cells, which occurs in various conditions, causes a reduction in the cochlear sensitivity by about 50 dB and the alteration of frequency discrimination. Total RNA was extracted from about 2000 mechanically dissociated OHCs, and a polymerase chain reaction (PCR) amplified cDNA library was constructed. The presence of the alpha-9 acetylcholine receptor subunit, preferentially expressed in OHCs, was found by direct PCR amplification of the library. A systematic sequencing of 218 clones showed 78% known genes, 11% EST-related sequences, and 11% unknown genes. The known-gene group was characterized by two main features: a large proportion (55%) of mitochondrial transcripts and an abundance in calcium-binding proteins, such as calmodulin and calbindin, for which expression has already been demonstrated in OHCs. Another protein, the oncomodulin recently shown to be OHC specific, was also found, and its mRNA expression was confirmed by in situ hybridization. Among the 24 unknown genes, 7 were expressed in a restricted pattern, including one expressed in cochlea and spleen and, to a lesser extent, in lungs.

Fibroblast growth factor receptor expression in outer hair cells of rat cochlea.

Fibroblast growth factors (FGFs) are critical for normal development of the organ of Corti, and may also protect hair cells from ototoxic damage. Four different fibroblast growth factors are known, three of which have different splice variants in the extracellular immunoglobin-like (Ig) III FGF-binding domain, giving different patterns of sensitivity to the different FGFs. Analysis of a cDNA library of rat outer hair cells by the polymerase chain reaction, using isoform specific primers, showed expression only of FGF receptor 3, splice variant IIIc. This allows us to predict the pattern of sensitivity to applied FGFs, which may be useful in targeting outer hair cells selectively during an FGF-based strategy for cochlear therapy.

Identification of preferentially expressed cochlear genes by systematic sequencing of a rat cochlea cDNA library.

107 expressed sequence tags (ESTs) from a rat cochlea cDNA library were identified by systematic sequencing coupled to database selection and RT-PCR analysis of novel sequences. This approach led us to select a clone, pCO8, showing no significant homology with any database sequence, that corresponds to a mRNA whose expression is restricted to the cochlea, except for traces detected in brain. Additional clones with novel sequences enriched in the cochlea were also found. ESTs bearing significant homologies with database sequences (63 out of 107) were classified according to the putatively encoded protein. They include tissue-specific genes not previously described in the cochlea as well as known genes from other species. We performed in situ hybridization in cochlear tissues to localize the pCO8 mRNA and that of clone pCO6 which is 100% homologous to the delayed rectifier potassium channel drk1. We found that both mRNAs were exclusively expressed in the cellular body of the primary auditory neurons from the spiral ganglion of the cochlea. These results indicate that this approach is an efficient way to identify novel genes that could be of importance in cochlear function.

A simple technique to efficiently dissociate primary auditory neurons from 5 day-old rat cochleas.

The aim of this work was to develop a simple and reproducible method of dissociation of cochlear spiral ganglion neurons in the rat. This technique, wich was developed in 5 day-old rat pups, was based on the use of a single enzyme, thermolysin. It is easy to set up and allows the collection of a large amount of neurons. These isolated neurons were kept in a definite, serum free culture medium up to 7 days. Neurons were characterized both by standard morphological criteria and by using a specific neuronal marker (anti-neurofilament 200 kD) after 2 h and 7 days in culture. Cell viability, assessed by fluorescent dyes indicated that all isolated cells were healthy even after 7 days in vitro. The dissociation and culture methods were found very satisfactory and can be easily adapted to any kind of experiment requiring isolated spiral ganglion neurons.

A comparison of the effect of cochlear perfusion with ouabain on summating potentials and distortion product otoacoustic emissions in the guinea pig.

In order to investigate whether or not the summating potential (SP) and the 2f1-f2 distortion product otoacoustic emission (DPOAE) are due to related cochlear non-linearities, their behavior was studied in the guinea pig after intracochlear perfusion with ouabain and subsequent rinsing. The SP was evoked with either 4 or 8 kHz tone bursts, and the 2f1-f2 DPOAE was evoked with simultaneous presentations of 6.6 and 8 kHz continuous tones. After ouabain perfusion, DPOAE was dramatically reduced while the SP underwent only a small reduction. After rinsing out the ouabain with artificial perilymph, the DPOAE showed partial recovery while the SP displayed a large and long-lasting increase when compared to its a initial value. These results suggest that the non-linear processes giving rise to the SP and DPOAE are not identical.

Changes in 2f1-f2 distortion product otoacoustic emissions following alterations of cochlear metabolism.

This paper summarizes the results obtained from investigations in which distortion product otoacoustic emissions (DPOAEs) were studied together with other cochlear physiological parameters. The cochlear metabolism was subjected to three different experimental conditions: guinea pigs were either submitted to hypoxia, to an intra-cochlear perfusion of ouabain or to an intra-cochlear perfusion of naloxone. The data show that DPOAEs remain affected for a certain time after the metabolic perturbations were removed. The comparison of the behaviour of DPOAEs and of other cochlear parameters gives good indications on the way these different experimental procedures affect the functioning of the cochlea during and after their application.

Effect of reversible hypoxia on the compared time courses of endocochlear potential and 2f1-f2 distortion products.

In order to study the effects of a controlled hypoxia on the cochlear active mechanisms, the 2f1-f2 distortion product (DP) and the endocochlear potential (EP) were recorded simultaneously in the same ear, in guinea pigs artificially respired with gas mixtures containing different percentages of oxygen. The data show an important difference in the behavior of the two parameters. While the EP undergoes a reduction of amplitude starting shortly after the establishment of the hypoxia, reaches a steady state, and recovers monotonically after a return to normoxic conditions, the time course of the DP is more complex. Its level also declines shortly after the beginning of the hypoxia though it slightly lags behind the EP decline. After switching back to normoxic conditions, the DP rises with an eventual delay with respect to the EP, overshoots, and then dramatically falls again. A slow recovery subsequently takes place and normal values are reached within 5 to 10 min. These results indicate a certain independence of the DP versus the EP. During the exposure to hypoxic conditions, differences in the time course and in the variation of amplitude of the two recorded parameters seem to indicate that the DPs could be more related to the OHC physiology than to the EP. The DP post-hypoxia effect observed after a return to normoxic conditions, indicates that a normal EP is not sufficient for the generation of normal DPs. Different hypotheses which could explain the DP post-hypoxia effect are discussed.

Activation of muscarinic cholinergic receptors stimulates inositol phosphates synthesis in the developing avian cochlear duct.

We previously reported that the inositol phosphates (IPs) synthesis is induced by muscarinic agonists in the rat cochlea and that this stimulation is maximal at postnatal day 12. This peak response is concomitant with the onset of the efferent synaptogenesis at the outer hair cell level. Whether the correlation between this neuronal plasticity and the enhanced IPs formation is unique to the rat or a general feature of the developing vertebrate cochlea is not known. To examine this question, we measured, in the presence of LiCl, the accumulation of (3H)-IPs induced by carbachol, in the developing chick cochlear duct during a period ranging from embryonic day (E) 8 to post-hatching day (P) 20. Carbachol (1 mM) causes a significant increase of IPs formation relative to basal values at all ages. This IPs accumulation is maximal at E8 (1854% of the basal level), then, rapidly decreases until P13 when it reaches a steady-state level of 294% of the basal level. Strikingly, this gradual decline in IPs formation is interrupted between E15 and E19, by a transient increase in IPs synthesis. This rise peaks at E16 with a stimulation value of 757% of the control level. This maximal stimulation is inhibited by atropine in a dose-dependent manner, as is the case at E9, suggesting the involvement of muscarinic receptors. Interestingly, the occurrence of the peak response is concomitant with the plastic events associated with the maturation of the efferent innervation of the cochlear duct. Thus, these results suggest that there may be a correlation between cochlear plasticity and enhanced IPs synthesis, which is not species-specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Cochlear origin of 2f1-f2 distortion products assessed by using 2 types of mutant mice.

Mutant mice with a particular type of cochlear pathology are excellent models to study the functional role of various structures in the cochlea. In order to assess the contribution of inner and outer hair cells to the generation of distortion product emissions (DPEs) we have recorded the 2f1-f2 DPE in a control group of CBA mice, which have normal numbers of inner and outer hair cells and two different types of mutant mice: the Bronx-waltzer mice and the Wv/Wv mice. In the Bronx waltzer mutant mice, 70% of inner hair cells are missing whereas the outer hair cells are present in normal number. The distortion product emissions 2f1-f2 is clearly recordable with a 10-20 dB lower magnitude as compared to normal CBA control mice. The homozygous Wv/Wv mutant mice on the other hand present a selective outer hair cell loss as a constant defect with no progressive degeneration of the organ of Corti and an essentially normal inner hair cell population. The cubic distortion products 2f1-f2 could not be detected in all but one animal. Therefore, the present study strongly suggests, that the outer hair cells are critically involved in the production of DPEs.

Effect of contralateral sound stimulation on the distortion product 2F1-F2: evidence that the medial efferent system is involved.

The mechanical nonlinearity of the cochlea that is associated with normal cochlear function, induces distortion products that can be recorded in the external auditory canal. The acoustic cubic distortion product 2F1-F2 (DP) was measured in the external canal in the presence and in the absence of a contralateral white noise. The experiments were carried out on 20 guinea pigs after a section of the middle ear muscles. They showed that the presence of a contralateral white noise induces a significant reversible reduction of the magnitude of the DPs. This suppressive effect produced by the contralateral white noise was completely canceled out by the midline sagittal section of the brainstem. This report supports the hypothesis that the suppressive effect of a contralateral sound stimulation is mediated by the medial efferent system.

Glutamate neurotoxicity in the cochlea: a possible consequence of ischaemic or anoxic conditions occurring in ageing.

Glutamate is considered to be one of the most common neurotransmitters in the fast excitatory synapses in the central nervous system. On the other hand, its excitotoxic properties are increasingly cited to explain some of the brain damage linked with hypoxia and ischaemia: i.e., those that occur frequently in ageing. An excess release of glutamate could, either directly or indirectly, activate receptors on the postsynaptic neuron, causing ion influxes accompanied by a massive entry of water, which would lead to an acute swelling of dendrites. In addition, calcium influx deregulates calcium homeostasis, which could lead to cell death. In the cochlea, glutamate is now considered to be one of the best candidates to mediate neurotransmission between inner hair cells (IHCs) and the auditory nerve dendrites. Among the variety of anatomical and physiological findings supporting the glutamate hypothesis, is the striking similarity of acute damage in the organ of Corti caused by exposure to a glutamate analogue (kainic acid), or by hypoxia, or even by an intense loud noise. In all cases an immediate swelling is observed, specifically affecting the radial afferents below the IHCs. The best explanation for this swelling is related to glutamate (or glutamate analogue) excitotoxicity. Thus, some of the cochlear damage that occur with ageing, especially the loss of the radial afferent fibres and type I ganglion cells, might well be attributed to glutamate excitotoxicity linked to vascular atrophy. The present paper discusses this hypothesis.

Effects of in vivo perfusion of glutamate dehydrogenase in the guinea pig cochlea on the VIIIth nerve compound action potential.

Glutamate is considered as the best candidate for the neurotransmission between the inner hair cell and the primary efferent neurons in the mammalian cochlea. In order to test its presence in the synapse, a degradative enzyme for glutamate, glutamate dehydrogenase (GDH) was perfused in the cochlea of guinea pigs. The intensity function of the VIIIth nerve compound action potential was recorded as a physiological test. The results show that the GDH induces a decrease in the auditory nerve responsiveness. The threshold elevation observed is dependent upon the enzyme concentration.

Morphological transformation of hair cells in the chick basilar papilla following an acoustic overstimulation.

The anatomical damage occurring to the chick basilar papilla following an exposure to a 125 dB SPL pure tone has been studied using scanning and transmission electron microscopy. By combining these two techniques it was possible to describe in detail certain types of damage occurring to hair cells at the periphery of the traumatized area. Abnormalities such as (1) hair cells with a 'giant' apical surface, or (2) with a small apical surface, or (3) without a cuticular plate probably represent stages of hair cell dedifferentiation.

Effect of noise level on the Met-enkephalin content of the guinea pig cochlea.

Using a highly sensitive and specific radioimmunoassay for Met-enkephalin, we have monitored in two series of experiments the changes of the Met-enkephalin content of guinea pig cochleas following a 60 min exposure to different intensities of white noise (70 dB SPL, 90 dB SPL, 110 dB SPL). Our results indicate that the Met-enkephalin content was significantly lower after noise exposures than after exposure to the silence of a sound attenuated chamber. After a stimulation at 70 dB SPL, the levels of Met-enkephalin were 70% (series I) and 61% (series II) of those obtained after a period of silence. After a 110 dB SPL stimulation, these values fell to 41% (series I) and 55% (series II) of those in silence. These results strengthen the hypothesis that enkephalins are olivocochlear neuroactive substances. They suggest that the enkephalin-containing lateral olivocochlear system discharges with noise stimuli of moderate intensity.

[Oto-acoustic emissions. II. Spontaneous oto-emissions: results in normal subjects or patients with tinnitus]. / Les oto-émissions acoustiques. II. Les oto-émissions spontanées: résultats chez des sujets normaux ou présentant des acouphènes.

Sounds can be emitted by the cochlea in the absence of any stimulation. This phenomenon is called spontaneous oto-acoustic emissions (SOAEs) and they can be recorded in the external auditory canal using a sensitive microphone. This phenomenon seems to be due to an abnormality of the active cochlear mechanisms. SOAEs have been studied in 140 human subjects, 140 of them suffering with tinnitus, while 80 were used as the control group. SOAEs have been studied as a function of age, and of the audiometric state. Possible relationships between SOAEs and tinnitus have also been studied. This study shows the interest of recording SOAEs for an early diagnosis of cochlear dysfunction. It also shows that there is no clear correlation between tinnitus and SOAEs.

Age-dependent effects of a pure tone trauma in the chick basilar papilla: evidence for a development of the tonotopic organization.

58 chicks or chick embryos were continuously exposed during 12 h on either embryonic day 18 or 20 or on post-hatching days 1, 10, 20 or 30 to 1.5 kHz pure tone at an intensity of 125 dB SPL. After a 20- or 30-day survival time, audiograms were recorded and then the basilar papillae were prepared for scanning electron microscopy. The frequency of maximum threshold elevation was seen at about 4 kHz when the chicks were exposed to the traumatic tone at embryonic day 18. It was shifted toward lower frequencies when the exposure was done at later stages. This shifting ended when the animals were exposed one day after hatching. After this stage, the maximum threshold elevation stabilized about one octave above the frequency of the traumatic tone. The position of maximum anatomical damage was located at 29.14% of the total length of the basilar papilla measured from the base when the exposure was done at embryonic day 20. It was shifted to 37% when the chicks were exposed one day after hatching or later. These results are in good agreement with recent hypotheses on development of the place principle. This development change seems to end at post-natal day 1 which also corresponds to the end of the anatomical and functional maturation of the basilar papilla.