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.

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.

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.

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.

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.

Electrophysiological study of the maturation of auditory responses from the inner ear of the chick.

Three electrophysiological functions of the chick basilar papilla were studied during development by recording the compound actin potential (AP) at the round window. The auditory thresholds showed a continuous maturation between the fifteenth day of incubation (E15) and the first post-hatching day (P1), when they attained adult values. Responses matured first to low frequencies and later to high frequency stimuli. The input-output (intensity-amplitude) functions matured regularly and never demonstrated the classical two slopes seen in mammals. The tuning properties, studied by tone-on-tone masking of the AP, achieved mature values before the thresholds; the Q10s reached adult values at E17 for a 500-Hz probe tone and at E19 for a 1000-Hz probe tone. The fact that a low-to-high frequency development trend was found with the embryonic middle ear cleared of fluids further suggests that this property of auditory ontogeny may be a function of changes in the transduction properties of the cochlea.

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.

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)

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.

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.

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.

Variability of the hereditary deafness in the white cat. I. Physiology.

Electrophysiological investigations, at different levels of the auditory pathway, were performed on 54 white cats. Hearing tests generally consisted of recording electrocochleograms or making audiograms from collicular or cortical responses. Some investigations were performed with chronically implanted electrodes to detect the first appearance of a hearing defect. Results showed no evident relationship between the age of the white cat and the appearance, severity or completeness of hearing loss. A large variety of hearing remnants was encountered in partly deaf animals which is tentatively related to a similar variety of histological damage of the cochlea described in a companion paper (Rebillard, M., Pujol, R. and Rebillard, G. (1981): Hearing Res. 5, 189-200).

Variability of the hereditary deafness in the white cat. II. Histology.

Cochlear degeneration in white cats was investigated by light- and electron-microscopic examinations. A great variety of histological damage was encountered both in completely and partially deaf animals. These variable features are discussed mainly with regard to the rate of degeneration and the site of the first damage. Atypical findings, such as primary degeneration of spiral ganglion neurons, are presented. The possibility for some damaged cochleas to work without hair cells is described. These results clearly demonstrate that hereditary degeneration in the cochlea of white cats is not a unique and regular process.

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.

Primary neural disorders in the deaf white cat cochlea.

Eleven white kittens were investigated from 2 days after birth up to an age of 4 months. After their hearing ability or deafness had been tested electrophysiologically, electron microscopic work was done at the level of the cochlea. Previous histological data indicated that the hereditary process of degeneration begins at the epithelial and sensory elements of the cochlea, and that the neural degeneration is only a secondary and very slow process. Results presented here indicate that, at least in some white cats, this assumption needs to be modified. Early failure in the myelination of the lamina spiralis fibres was noticed in most of the white kittens. Furthermore, two kittens (7 and 16 days old) presented a complete degeneration of the spiral ganglion neurons, and signs of anterograde degeneration of their fibres going to the organ of Corti. These primary neural defects indicate that hereditary hearing defects may directly affect both epithelio-sensory and neural structures.

Histo-physiological relationships in the deaf white cat auditory system.

An investigation is underway in deaf white cats in order to clear up some problems about the timing of cochlear degeneration, the relationships between physiological and histological findings, and the consequential state of central auditory pathways. The first results reported here show that degeneration of sensory structures was much more advanced than physiological results lead us to believe. The possibility of direct fibre stimulation is discussed. Although degeneration of nervous structures was delayed compared with sensory ones, some early abnormalities in myelination were found. In the adult, completely deaf for years, the electrical stimulation of cochlear nuclei evoked normal-looking field responses at the cortical level. Thus some degree of auditory pathway functional integrity appeared even long after they were disconnected from receptors.

[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.