[Endolymph homeostasis and Menière's disease: fundamentals, pathological changes, aminoglycosides]. / Endolymph-Homöostase und M. Menière : Grundlagen, pathologische Veränderungen, Aminoglykoside.

Although low dose intratympanal gentamicin has empirically been very effective in treating Menière's disease, the mechanisms of elimination or amelioration of vertigo are still insufficiently understood. Most animal studies investigating the effect of aminoglycosides used high doses that damage or kill hair cells and many other cell types of the inner ear. Additional studies are needed to investigate the effects of low dose gentamicin to elucidate the mechanisms affecting vertigo. In this article it will be explained how disturbances of endolymph homeostasis lead to endolymphatic hydrops and finally to leakage of K(+) from the endolymph into the perilymphatic space. This can lead to a non-physiological activation of vestibular nerve fibres thus causing vertigo.

Safety of gold in stapes surgery.

Gold prostheses in middle ear surgery were found to have a higher extrusion rate than prostheses made from titanium. Incidences of deafness after insertion of a gold piston into the vestibule during stapes surgery have been observed. The aim of this study was to find out to what degree gold cations can diffuse from the prosthesis piston into the perilymph. For this, gold prostheses were incubated in artificial perilymph for four months, after which the gold content of the perilymph was analysed. As gold exhibits a special behaviour in complex fluids like the perilymph, a new analysing method was developed. The results show that gold does leak out of the pistons, that it can be reliably measured and that the amount of gold reaching the perilymph depends on the contact area. As the amount of gold measured in the perilymph stays far below the toxic level, it is very unlikely that the gold cations diffusing from a stapes prosthesis into the perilymph have a toxic effect on the inner ear hair cells. Inflammatory or allergic reactions to gold induced by direct tissue contact, however, cannot be excluded.

Evidence for supporting cell proliferation and hair cell differentiation in the basilar papilla of adult Belgian Waterslager canaries (Serinus canarius).

We used the bromodeoxyuridine technique to study the proliferative activity in the basilar papilla of normal and Belgian Waterslager canaries with and without preceding sound trauma. Without sound trauma, there were, on average, six supporting cell divisions per day in the basilar papilla of Waterslager canaries. This rate of supporting cell proliferation corresponds well with estimates of the rate of hair cell differentiation derived from counts of immature-appearing hair cells obtained by using scanning electron microscopy of the Waterslager basilar papilla. Thus, supporting cell division appeared correlated with hair cell differentiation in Waterslager canaries. Bromodeoxyuridine labeling of cells in undamaged non-Waterslager canaries also indicated a very low rate of supporting cell division. In contrast with Waterslager canaries, this low rate of proliferation was not associated with a measurable rate of hair cell differentiation. In both normal and Waterslager canaries, exposure to traumatizing sound induced a dramatic increase in the rate of cell proliferation. These data show that a very low rate of supporting cell proliferation is normally present in birds, but it is not associated with a corresponding rate differentiation of hair cells. Only an increase above this low ambient rate of supporting cell proliferation, such as that following loss of hair cells, induces the differentiation of new hair cells in birds. The reason why Waterslager canaries do not completely compensate for their inherited hair cell deficit of 30% is not clear, when they can clearly respond to additional cochlear trauma from noise exposure with an increase in proliferation rate.

Domestication differentially affects cochlear nucleus subdivisions in the gerbil.

We analyzed the effects of domestication on the subdivisions of the cochlear nucleus in the gerbil (Meriones unguiculatus) by comparing their volumes and rostrocaudal extents in laboratory gerbils and in age-matched F1 offspring of gerbils caught in the wild. In addition, soma size was systematically analyzed in the anteroventral cochlear nucleus of both groups. Total cochlear nucleus volume and rostrocaudal extent were not significantly different between groups either for young (postnatal day 9) animals before the onset of hearing or for young 4-month-old animals. However, the dorsal cochlear nucleus was significantly larger and the anteroventral cochlear nucleus was significantly smaller in young adults of the wild strain. Thus the relative proportions of the cochlear nucleus subdivisions differed between the groups. In addition, soma size was significantly larger in the low-frequency portion of the anterovental cochlear nucleus in domesticated gerbils compared to wild gerbils. To our knowledge, this is the first reported instance of a well-defined brain structure (e.g., the antreovental cochlear nucleus) being larger in the domesticated than in the wild form.

Sound induced expression of c-Fos in GABA positive neurones of the gerbil cochlear nucleus.

To characterize c-Fos-expressing neurones in more detail, a double labelling technique was developed to simultaneously demonstrate the presence of GABA-like and c-Fos-like immunoreactivity in cryostat sections of the gerbil cochlear nucleus. The animals were exposed to a wide-band noise stimulus for the induction of c-Fos expression. The distribution of c-Fos-positive cells and GABA-positive somata was consistent with previous reports. Our double labelling approach revealed the presence of GABA-positive cells that showed c-Fos expression after noise stimulation. One interpretation of this finding is that a prolonged acoustic exposure induces long term changes in inhibitory interneurones to adjust the output of the cochlear nucleus.

Auditory primary afferents in the starling: correlation of function and morphology.

Despite the independent evolution of birds and mammals, a number of structural similarities of their hearing organs have developed in parallel. By tracing the peripheral origin of functionally-characterized primary neurons, the present study demonstrates functional similarities between the respective hair cell populations of the hearing organs of birds and mammals. The space devoted to one octave on the starling's basilar papilla is not constant over the whole length; rather it increases from the apical low- to the basal high-frequency end. The finding that (with the exception of a specialized area near the apical end) only tall hair cells situated on the neural limbus receive active afferent innervation is a functional parallel to the mammalian inner hair cells. The thresholds of afferents increase with distance of the related hair cells from the neural side of the papilla and cover a range of more than 50 dB within the area of tall hair cells.

The distribution of N-acetylgalactosamine in the cochlear nucleus of the gerbil revealed by lectin binding with soybean agglutinin.

A horseradish peroxidase conjugated lectin from Glycine max (soy bean agglutinin; SBA) was used to characterise the distribution of N-acetylgalactosamine in the cochlear nucleus of the mongolian gerbil. SBA bound differentially to a variety of structures within the cochlear nucleus. Specific SBA labelling was associated with large non-granule neurones of variable size and shape throughout the cochlear nucleus. Compared to adjacent Nissl-stained sections 80% of the non-granule cells in the dorsal cochlear nucleus (DCN) and more than 90% of the non-granule cells in the ventral cochlear nucleus (VCN) bound SBA. The variation in location, size and shape as well as the high percentage of the labelled neurones suggest that cells of several, if not all, non-granule cell types, which have been described for the cochlear nucleus according to the usual Nissl schemes, are SBA positive. Granule cells did not bind SBA because all SBA-labelled cells had diameters above 10 microns. Diffuse labelling, not systematically associated with cells or fibres, was high in the molecular and fusiform cell layers of the DCN and that part of the granule cell area located close to the surface of the VCN. Darkly labelled granules (up to 2 microns diameter) were prominent in the area of the VIIIth nerve root. After long SBA incubations, they were also present in VCN and to a lesser degree in DCN. The results are discussed with respect to findings in other brain areas and the possible co-localisation of gamma aminobutyric acid (GABA), parvalbumin and N-acetylgalactosamine.

The diameters of guinea pig auditory nerve fibres: distribution and correlation with spontaneous rate.

In the mammalian auditory nerve physiological recordings revealed that the spontaneous discharge rate of single auditory fibres correlates with the diversity of input-output functions which may be important for intensity discrimination (e.g., Sachs and Abbas, 1974, Liberman, 1978; Winter et al., 1990). In this study we determined if the spontaneous discharge rate of auditory nerve fibres in the guinea pig is correlated with an anatomical feature, namely the diameter of the respective fibres. The diameter of myelinated (Type I) guinea pig auditory nerve fibres was measured after staining with different techniques. Measurements were made on semithin sections using a video image analysis system. The diameters of fibres stained with toluidine blue from the portion of the auditory nerve containing fibres from the basal turn of the cochlea were found to have a normal distribution. Fibres were also labelled with horseradish peroxidase by bulk injection into the spiral ganglion. It was found that the presence of horseradish peroxidase within the fibres reduced the measured diameter in comparison to adjacent unlabelled fibres. A number of fibres were physiologically characterized with respect to spontaneous discharge rate and subsequently intracellularly labelled with horseradish peroxidase. Fibre diameter of a selected sample of intracellularly fibres was measured over a distance of 800 microns within the internal auditory meatus. At the positions nearest to the spiral ganglion fibres possessing low spontaneous rates were found to have smaller diameters than high spontaneous rate fibres. No difference in fibre diameter was found for the positions near the cochlear nucleus.

Cobalt labelling of single primary auditory neurones: an alternative to HRP.

We have labelled single, primary auditory neurones in three reptile and one bird species. After functional characterization of the neurones, hexamminecobaltic chloride was iontophoretically injected through the recording micropipette. Precipitation of cobalt sulfide followed by silver intensification of the cochlear duct as a whole-mount preparation revealed stained neurones in over 90% of cases. This method has several advantages over labelling with HRP.

Quantitative morphological analysis of the sensory epithelium of the starling and pigeon basilar papilla.

Several features of bird basilar papilla morphology were quantitatively studied in the starling and the pigeon in order to attempt a structure-function correlation. We confirmed and quantified several findings from earlier studies, but also obtained results contradictory to previous reports. The greatest discrepancies concerned the pattern of hair cell orientation. By including the results from other investigations, we describe a 'typical' avian basilar papilla and on this basis the specializations within individual species. These morphological specializations are discussed in the context of the available physiological data.

The phase response of primary auditory afferents in a songbird (Sturnus vulgaris L.).

The effects of stimulus frequency and intensity on phase-locking characteristics of cochlear ganglion cells were studied in the starling. All cells showed phase-locking to tone stimuli within their response area. Phase-locking at CF is found on average 9 dB below discharge rate threshold. Phase-locking is best at 0.4 kHz and deteriorates with increasing frequency almost independently of CF. No phase-locking was evident for test frequencies above 3-4 kHz. Phase-locking in cells with CFs above 1.0 kHz is better below CF than at CF. For constant sound pressure, an increase in stimulus frequency always produced an increase in phase lag of the neural response. The phase vs. frequency data obtained at constant sound pressure can be reasonably approximated by straight line functions. The slopes of these functions indicate the latency of the neural response, and are correlated with the CFs of the respective cells; the latency tends to be longer in low-CF cells and shorter in high-CF cells. The latency decreases by 0.04 ms per 1 dB sound pressure increase. The response phase at CF is nearly stimulus level-independent. Increasing stimulus intensity causes increasing phase lag below CF and decreasing phase lag above CF. These results are compared to findings in other vertebrates and demonstrate the similarities of phase-locking characteristics despite the substantial anatomical differences among the vertebrate groups.

Temporal modulation transfer functions in the European Starling (Sturnus vulgaris): II. Responses of auditory-nerve fibres.

The temporal resolution of cochlear-nerve fibres in the European starling was determined with sinusoidally amplitude-modulated noise stimuli similar to those previously used in a psychoacoustic study in this species (Klump and Okanoya, 1991). Temporal modulation transfer curves (TMTFs) were constructed for cochlear afferents allowing a direct comparison with the starling's behavioural performance. On average, the neuron's detection of modulation was less sensitive than that obtained in the behavioural experiments, although the most sensitive cells approached the values determined psychophysically. The shapes of the neural TMTFs generally resembled low-pass or band-pass filter functions, and the shapes of the averaged neural functions were very similar to those obtained in the behavioural study for two different types of stimuli (gated and continuous carrier). Minimum integration times calculated from the upper cut-off frequency of the neural TMTFs had a median of 0.97 ms with a range of 0.25 to 15.9 ms. The relations between the minimum integration times and the tuning characteristics of the cells (tuning curve bandwidth, Q10 dB-value, high- and low-frequency slopes of the tuning curves) are discussed. Finally, we compare the TMTF data recorded in the starling auditory nerve with data from neurophysiological and behavioural observations on temporal resolution using other experimental paradigms in this and other vertebrate species.

Effects of L-glutamate on auditory afferent activity in view of its proposed excitatory transmitter role in the mammalian cochlea.

This report describes the effects of L-glutamate (Glu) introduced into the perilymph of scala tympani on the spontaneous and tone-evoked activity of guinea pig single primary auditory afferents. Concentrations below 2 mmol/l were in general ineffective, while a concentration of 5 mmol/l caused a marked decrease of the neural activity. At 2 mmol/l, roughly 60% of the Glu-perfusions were effective and produced a variety of changes. The most prominent effect was a reduction of the tone-evoked activity without a change in spontaneous rate. Indeed, in some cells, the tone-evoked activity could be almost totally abolished without affecting the spontaneous activity. More rarely observed was a moderate, generally transient increase of the spontaneous activity which was occasionally followed by a decrease in both tone-evoked and spontaneous firing rate. The increase in firing rate was always small relative to the maximum discharge rate evoked by tone stimuli. Desensitization of the Glu-evoked response without an obvious change in the spontaneous activity was also found. In a few cells Glu caused a reduction of the discharge rate below the spontaneous firing rate during loud tone presentation. Higher Glu doses generally caused a reduction of spontaneous and tone-evoked activity without any sign of a preceding increase. Thus, the effects of Glu in the mammalian cochlea appear to be complex and on balance seem inconsistent with the effects predicted for an excitatory transmitter. The findings argue against the hypothesis that Glu is the afferent transmitter released by inner hair-cells. However, the results do not exclude an involvement of Glu as a neuromodulator or co-transmitter.

Neither endocochlear potential nor tegmentum vasculosum are affected in hearing impaired belgian waterslager canaries.

We previously showed that the Belgian Waterslager canary strain is affected by a hereditary hearing loss that is associated with a reduced number of hair cells and hair cell pathologies in the basilar papilla. Since hair cell pathologies were also present in the sacculus, Weisleder et al. (1994) suggested that these birds are afflicted by Scheibe's like dysplasia, a cochleo-saccular defect. In mammals, cochleo-saccular defects are characterized primarily by the lack of an endocochlear potential and abnormalities in the Stria vascularis which only secondarily lead to hair cell loss (Steel and Bock, 1983; Steel, 1994; 1995). Here we report the endocochlear potential of six ears from three non-Belgian Waterslager canaries and three ears of two Belgian Waterslager canaries to decide if Waterslager canaries are affected by a cochleo-saccular or by a neuroepithelial defect. The mean endocochlear potential was 17.6+/-2. 5 mV in the non-Waterslager canaries and 20.3+/-0.6 mV in Waterslager canaries. In addition, and consistent with the presence of a normal endocochlear potential, light microscopy of the tegmentum vasculosum provided no evidence for pathology. These data show that Belgian Waterslager canaries are affected by a neuroepithelial rather than a cochleo-saccular inner ear defect.

A quantitative analysis of the nerve fibers in the VIIIth nerve of Belgian Waterslager canaries with a hereditary sensorineural hearing loss.

The number of auditory nerve fibers was determined for non-Belgian Waterslager canaries (non-BWS) and Belgian Waterslager canaries (BWS) that are affected by a sensorineural high frequency hearing loss and a 30% reduction in the number of auditory hair cells. Counts were obtained from semithin cross sections of the Durcupan-embedded auditory nerve at the level of the internal auditory meatus. In addition, the number of lagenar fibers was determined from cross sections near the apical end of the cochlear duct in order to separate them from the total number of auditory nerve fibers. The mean number of auditory nerve fibers was 6076 in non-BWS and 5363 in BWS canaries, representing a 12% reduction in BWS. This small reduction in the number of auditory nerve fibers, as compared to the larger reduction in hair cell number, might be explained by a predominant loss of abneural hair cells in BWS, since it has been shown for other species that a large proportion of abneural hair cells are devoid of afferent innervation. In addition, we observed that despite the prominent hair cell pathologies documented for BWS canaries, the mean diameter of auditory nerve fibers from non-BWS canaries (2.22+/-0.81 microm) did not differ from those of BWS canaries (2.21+/-0.96 microm).

Inner-ear abnormalities and their functional consequences in Belgian Waterslager canaries (Serinus canarius).

Recent reports of elevated auditory thresholds in canaries of the Belgian Waterslager strain have shown that this strain has an inherited auditory deficit in which absolute auditory thresholds at high frequencies (i.e. above 2.0 kHz) are as much as 40 dB less sensitive than the thresholds of mixed-breed canaries and those of other strains. The measurement of CAP audiograms showed that the hearing deficit is already present at the level of the auditory nerve (Gleich and Dooling, 1992). Here we show gross abnormalities in the anatomy of the basilar papilla of Belgian Waterslager canaries at the level of the hair cell. The extent of these abnormalities was correlated with the amount of hearing deficit as measured behaviorally.

Peripheral basis for the auditory deficit in Belgian Waterslager canaries (Serinus canarius).

Recently, behavioural thresholds obtained in canaries of the Belgian Waterslager strain showed that these birds have an inherited auditory deficit. Canaries of this strain have absolute auditory thresholds at frequencies above 2.0 kHz that are as much as 40 dB above the threshold of canaries of other strains. We obtained audiograms from cochlear microphonics and from compound action potentials from the 8th nerve of Waterslager and non-Waterslager canaries and compare these results to previous behavioural data on hearing in this species. We also examined the growth of evoked potential amplitude-intensity functions in Waterslager and non-Waterslager canaries. Together with reflectance measurements of middle-ear function from both Waterslager and non-Waterslager canaries, we conclude that the origin of auditory deficit in Waterslager canaries lies in the cochlea.

Fine structure of the basilar papilla of the emu: implications for the evolution of avian hair-cell types.

The morphology of the basilar papilla of the emu was investigated quantitatively with light and scanning electron microscopical techniques. The emu is a member of the Paleognathae, a group of flightless birds that represent the most primitive living avian species. The comparison of the emu papilla with that of other, more advanced birds provides insights into the evolution of the avian papilla. The morphology of the emu papilla is that of an unspecialised bird, but shows the full range of features previously shown to be typical for the avian basilar papilla. For example, the orientation of the hair cells' sensitive axes varied in characteristic fashion both along and across the papilla. Many of the quantitative details correlate well with the representation of predominantly low frequencies along the papilla. The most distinctive features were an unusually high density of hair cells and an unusual tallness of the hair-cell bodies. This suggests that the evolution of morphologically very short hair cells, which are a hallmark of avian papillae, is a recent development in evolution. The small degree of differentiation in hair-cell size contrasts with the observation that a significant number of hair cells in the emu lack afferent innervation. It is therefore suggested that the development of functionally different hair-cell types in birds preceded the differentiation into morphologically tall and short hair cells.

Activity patterns of primary auditory-nerve fibres in chickens: development of fundamental properties.

We have examined the activity patterns of single auditory-nerve fibers in the chicken and tested for possible changes during post-hatching development. For this purpose, we recorded from fibres in the cochlear ganglion of chickens of two age groups (about P2 and P21) and investigated their spontaneous and sound-evoked activity patterns under nembutal-chloralhydrate anaesthesia. The spontaneous activity of primary auditory neurones was irregular, the average rates were between 20.5 (P2) and 23 (P21) spikes/s. Many low-frequency fibres from both age groups showed preferred intervals in their spontaneous activity. Tuning characteristics, including the range of characteristic frequencies, the presence of primary and two-tone suppression, the slopes of tuning-curve flanks and Q10dB values were similar to those previously reported for the starling and were statistically indistinguishable between the two age groups. However, there was a difference in fibre thresholds at the highest frequencies. Systematic differences were also present between the two age groups with regard to some characteristics of the rate-intensity functions. These data indicate that whereas the tuning properties of primary auditory fibres of the chicken cochlea are mature as early as post-hatching day 2, the intensity functions are not.

Masking by harmonic complexes in birds: behavioral thresholds and cochlear responses.

Thresholds for pure tones embedded in harmonic complexes were measured behaviorally and physiologically for three species of birds, and physiologically in gerbils. The harmonic maskers were generated using the Schroeder-phase algorithm, characterized by monotonically increasing or decreasing phase across frequency. Previous work has shown that these stimuli produce large differences in masking in humans but not budgerigars. In this study, we show that for two additional species of birds, the patterns of masking were similar to those shown for budgerigars, with masking differing only slightly for the two Schroeder-phase waveforms, and in the opposite direction from that demonstrated in humans. Amounts of masking among species corresponded qualitatively to differences in their critical ratios. Evoked potential measurements in birds and gerbils indicated responses that were consistent with the behaviorally measured thresholds in birds and humans. Results are interpreted in light of differences in frequency selectivity and cochlear temporal processing across species.