Supporting cells contribute to control of hearing sensitivity.

The mammalian hearing organ, the organ of Corti, was studied in an in vitro preparation of the guinea pig temporal bone. As in vivo, the hearing organ responded with an electrical potential, the cochlear microphonic potential, when stimulated with a test tone. After exposure to intense sound, the response to the test tone was reduced. The electrical response either recovered within 10-20 min or remained permanently reduced, thus corresponding to a temporary or sustained loss of sensitivity. Using laser scanning confocal microscopy, stimulus-induced changes of the cellular structure of the hearing organ were simultaneously studied. The cells in the organ were labeled with two fluorescent probes, a membrane dye and a cytoplasm dye, showing enzymatic activity in living cells. Confocal microscopy images were collected and compared before and after intense sound exposure. The results were as follows. (1) The organ of Corti could be divided into two different structural entities in terms of their susceptibility to damage: an inner, structurally stable region comprised of the inner hair cell with its supporting cells and the inner and outer pillar cells; and an outer region that exhibited dynamic structural changes and consisted of the outer hair cells and the third Deiters' cell with its attached Hensen's cells. (2) Exposure to intense sound caused the Deiters' cells and Hensen's cells to move in toward the center of the cochlear turn. (3) This event coincided with a reduced sensitivity to the test tone (i.e., reduced cochlear microphonic potential). (4) The displacement and sensitivity loss could be reversible. It is concluded that these observations have relevance for understanding the mechanisms behind hearing loss after noise exposure and that the supporting cells take an active part in protection against trauma during high-intensity sound exposure.

The cellular localization of the neuropeptides substance P, neurokinin A, calcitonin gene-related peptide and neuropeptide Y in guinea-pig vestibular sensory organs: a high-resolution confocal microscopy study.

Four neuropeptides, substance P, neurokinin A, calcitonin gene-related peptide and neuropeptide Y, were detected by radioimmunoassay in guinea-pig vestibular end-organs. High-resolution confocal microscopy visualization of immunofluorescence staining was used to determine the cellular localization of these peptides. Substance P- and neurokinin A-like immunoreactivities were found to co-exist in afferent fibers innervating the peripheral regions of both the utricular and ampullar sensory organs. The immunoreactivity was more concentrated in the distal ends of the calyceal-shaped nerve endings that innervate type I sensory cells. While in the guinea-pig, nerve calyces and type I cells are distributed in both the central and peripheral regions of the sensory epithelia, immunoreactive calyces were found only in the peripheral regions. Calcitonin gene-related peptide-like immunoreactivity was localized in small bouton endings situated at the level of the base of the hair cells. These boutons were in a position to make axosomatic contacts with type II sensory cells and axodendritic contacts with afferent nerve endings. Calcitonin gene-related peptide immunoreactivity co-existed with choline acetyltransferase immunoreactivity. The localization and shape of these boutons identified them as the axonal endings of efferent vestibular fibers. Neuropeptide Y-like immunoreactivity was not observed in the actual sensory epithelium but in the underlying connective tissue, where it was located in varicose fibers along blood vessels. The synaptic position of the tachykinins is clearly distinct from that of calcitonin gene-related peptide. This segregation distinguishes the vestibular end-organs from most peripheral tissues where these peptides are co-localized. The tachykinin-immunoreactive afferent fibers are postsynaptic to the hair cells. If, as in somatic sensory endings, these fibers can be triggered to release the neuropeptides by an axon reflex type of activation, then the tachykinins could interfere directly with the function of type I and type II vestibular hair cells. Calcitonin gene-related peptide co-exists with acetylcholine in the efferent axonal endings that are presynaptic to type II hair cells and to afferent fibers. Calcitonin gene-related peptide can thus interfere by direct synaptic action with type II hair cells only. It may also regulate the activity of the tachykinin-containing afferents.

Vital staining of the hearing organ: visualization of cellular structure with confocal microscopy.

Cells inside the intact organ of Corti were labelled with fluorescent probes reflecting various aspects of structure and function. The dyes were introduced into the perilymphatic space by perfusion of the scala tympani of the temporal bone from the guinea-pig maintained in isolation. The dyes were able to diffuse through the basilar membrane and into the organ of Corti where they were spontaneously absorbed by the sensory and supporting cells. Confocal microscopic observation was made through an opening in the apex of the cochlea. A number of different dyes were used; a carbocyanine dye which stains mitochondria; two styryl dyes which are absorbed by the cell membranes and calcein, a cytoplasmic marker that fluoresces in vital cells. Extracellular space was stained by a cell-impermeant Dextran fluorescein. The most striking finding was that the membrane dyes preferentially stained the sensory cells and neural elements whereas the staining of the supporting cells was faint. The cytoplasmic dye in general stained sensory and supporting cells to the same extent. By tilting the organ, a view could be obtained from the side like a radial section through the organ. Outer and inner hair cells with their sensory hairs, nerve fibres and nerve endings, especially under the inner hair cells, could be seen in profile. Introduction of a high molecular weight Dextran into the endolymphatic space outlined the tectorial membrane which was seen in negative contrast. The simultaneous perfusion with a membrane dye stained the hair cells and their sensory hairs. Merging of the two images gave the possibility to examine, in the living tissue, the cilia to tectorial membrane relationship. Of general interest is the finding that the membrane dyes preferentially stained the sensory and neural elements of the nervous system, represented here by the hair cells and nerve fibres of the inner ear.

Presence of synapsin I in afferent and efferent nerve endings of vestibular sensory epithelia.

The presence and localization of synapsin I were investigated in the cat vestibular epithelium, using a rabbit anti-synapsin I antibody. The staining was performed by immunofluorescence or by a peroxidase-anti-peroxidase technique. A strong immunoreactivity was observed with both methods. It appeared as spherical patches distributed in the lower part of the epithelium. This distribution pattern is very similar to that of the efferent synaptic endings which are filled with microvesicles. Elongated immunoreactive segments were also observed around some hair cells. They suggest the presence of synapsin I in the afferent sensory endings contacting the hair cells. This immunoreactivity occurs in early postnatal and adult stages.

Changes in choline acetyltransferase, glutamic acid decarboxylase, high-affinity glutamate uptake and dopaminergic activity induced by kainic acid lesion of the thalamostriatal neurons.

Kainic acid lesion of the 'centre médian'-parafascicular complex of the thalamus inducing a degeneration of the thalamostriatal neurons was followed by a decrease in choline acetyltransferase (ChAT) in the rostral part of the striatum in the rat. This decrease in ChAT was concomitant with an increase in glutamate decarboxylase, high-affinity glutamate uptake and apparent dopamine turnover. These results suggest that the thalamostriatal partly cholinergic input exerts a powerful control over GABAergic, glutamatergic and dopaminergic neurons in the basal ganglia.

Alpha-fodrin (brain spectrin) immunocytochemical localization in rat vestibular hair cells.

The presence of a spectrin-related protein in rat vestibular sensory receptors was demonstrated by immunocytochemistry and immunoblotting using affinity purified anti alpha-fodrin antibodies. Intense immunoreactivity was found in the apical pole of sensory hair cells where it seems to be concentrated in the cuticular plate. In contrast, alpha-fodrin immunoreactivity was absent from the stereocilia. We suggest that a spectrin-related protein participates in the organization of the cuticular plate of vestibular hair cells by cross-linking actin filaments as well as by anchoring the cuticular plate to the apical cell membrane.

Fodrin immunocytochemical localization in the striated organelles of the rat vestibular hair cells.

The immunocytochemical distribution of a spectrin-related protein, fodrin was studied at the electron microscopic level in the rat vestibular hair cells. As previously demonstrated [Scarfone et al., Neurosci. Lett. 93, 13-18, 1988], an intense immunoreactivity was found in the cuticular plates. We demonstrate furthermore, here, for the first time the association of fodrin immunoreactivity with the striated infracuticular structures called striated organelles (SO). Fodrin was found in striated structures clearly identified as SO in both Type I and Type II hair cells. SO were labelled regardless of their location, subcuticular or associated with the plasma membrane of the cells. We suggest that fodrin, as in the cuticular plate, could participate to the Ca2+ dependent cross-linking of the actin filaments of the striated organelles and could play a role in their interaction with the submembraneous cytoskeleton.

Structural relationships of the unfixed tectorial membrane.

Although the tectorial membrane has a key role in the function of the organ of Corti, its structural relationship within the cochlear partition is still not fully characterised. Being an acellular structure, the tectorial membrane is not readily stained with dyes and is thus difficult to visualise. We present here detailed observations of the unfixed tectorial membrane in an in vitro preparation of the guinea pig cochlea using confocal microscopy. By perfusing the fluid compartments within the cochlear partition with fluorochrome-conjugated dextran solutions, the tectorial membrane stood out against the bright background. The tectorial membrane was seen as a relatively loose structure as indicated by the dextran molecules being able to diffuse within its entire volume. There were, however, regions showing much less staining, demonstrating a heterogeneous organisation of the membrane. Especially Hensen's stripe and regions facing the outer hair cell bundles appeared more condensed. Whereas no connections between Hensen's stripe and the inner hair cell bundles could be observed, there was clearly a contact zone between the stripe and the reticular lamina inside of the inner hair cell.

Anaesthetics may change the shape of isolated type I hair cells.

Type I hair cells isolated from animals anaesthetised with barbiturates or ether were found to be shorter and to lack a prominent 'neck' region when compared to cells isolated from non-anaesthetised animals. Ketamine did not have this effect. The changes observed could have important implications for the physiology of inner ear receptors. These findings infer that care should be taken in the choice of anaesthetics used in studies on cells from the inner ear.

Synapsin I and Synaptophysin expression during ontogenesis of the mouse peripheral vestibular system.

Synapsin I and Synaptophysin are selectively localized in axonal endings of CNS neurons where they are associated with small synaptic vesicle membranes. The development of expression of these 2 proteins was studied by immunocytochemistry during ontogenesis of the peripheral vestibular system in the mouse. Both proteins are localized in vestibular ganglion neurons and in their peripheral sensory extensions as early as gestational day 14. While the entire periphery of these fibers is labeled during embryogenesis, both proteins are subject to relocation during the postnatal maturation of these fibers. In the mature vestibular receptors they disappear from the fibers themselves but are found concentrated in their intraepithelial endings and in the neuronal cell body. These observations show that the distribution pattern of Synapsin I and Synaptophysin in peripheral extensions of vestibular afferent neurons during development is identical to that described in axonal processes of CNS neurons. This suggests that the peripheral processes of the vestibular afferent neurons present structural and biochemical characteristics of axons. These characteristics are consistent with a bimodal sensory and secretory function of mature endings.

Laser scanning confocal microscopy of the hearing organ: fluorochrome-dependent cellular damage is seen after overexposure.

In order to combine laser confocal microscopy with physiological measurements, a number of conditions have to be met: the dye must not be toxic to the cells the laser light itself must not damage the cells; and the excitation of the fluorochrome during imaging must not generate products with toxic effects. We have investigated these conditions the hearing organ of the guinea pig. Two dyes were used, namely, calcein-AM, which is metabolized in vital cells to a fluorescent product in the cytoplasm, and a lipophilic membrane dye. The effect of the dyes on cell function was tested in the intact hearing organ, maintained in the isolated temporal bone, by measuring the electrophysiological potentials generated by the sensory cells in response to tone pulses. The loading of the cells with the dyes had no adverse effects. The effect of the laser beam was explored on isolated coils from the cochlea. In two preparations, the specimens viewed in the confocal system were fixed and processed for electron microscopy. Identified cells were followed before, during, and after laser exposure and could ultimately be examined at the ultrastructural level. Exposure to the laser beam did not cause damage in unstained cells, even at high intensities. In stained tissue, confocal microscopy could safely be performed at normal beam intensity without causing ultrastructural changes. At high intensities, about 100 times normal for 60 times as long, irradiation damage was seen that was selective in that the cells stained with the different dyes exhibited damage at the different sites corresponding to the subcellular location of the dyes. Cells stained with calcein showed lysis of mitochondria and loss of cytoplasmic matrix, whereas cells stained with the styryl membrane dye showed swelling of subsurface cisternae, contortion of the cell wall, and shrinkage. The styryl dyes, in particular, which selectively stain the sensory and neuronal cells in the organ of Corti, could be exploited for phototoxic use.

Neurofilament proteins form an annular superstructure in guinea-pig type I vestibular hair cells.

Neurofilaments, the neuron-specific intermediate filaments, are composed of three immunochemically distinct subunits: NF-L, NF-M and NF-H that can be either phosphorylated or unphosphorylated. In mammals, the distribution of these subunits has been described in vestibular ganglion neurons, but there are no reports on the presence of neurofilaments in vestibular hair cells. We investigated, by immunocytochemistry, neurofilaments in vestibular hair cells from rat and guinea-pig using antibodies against the three subunits and to dephosphorylated NF-H (clone SMI 32, recognizes also NF-M on immunoblots), on Vibratome sections of the vestibular end-organs and on isolated hair cells. Various immunostaining protocols were used, as appropriate for the method of observation: laser scanning confocal microscopy (immunofluorescence) and transmission electron microscopy (immunoperoxidase, pre-embedding technique). In rat and guinea-pig cristae and utricles, neurofilament immunoreactivity was observed in axons inside and below the sensory epithelia. In guinea-pig, in addition to this staining, intensely immunoreactive annular structures were found in the basal regions of hair cells. These rings were detected with anti-NF-L, -NF-M and -dephosphorylated NF-H/M antibodies, but not with anti-phosphorylation-independent NF-H. Ring-containing hair cells were present in all regions of the sensory epithelia but were more abundant in the peripheral areas. All levels of observation (Vibratome and thin sections, and isolated hair cells) showed that only the guinea-pig type I hair cells contained a neurofilament ring. High-resolution observations showed that the ring was located below the nucleus, often close to smooth endoplasmic reticulum and the cell membrane.

[Demonstration and localization of synapsin I in vestibular receptors in the cat: immunocytochemical study]. / Mise en évidence et localisation de la synapsine I dans les récepteurs vestibulaires chez le chat: étude immunocytochimique.

The presence and localization of synapsin I, a neuron-specific phosphoprotein, was investigated in the cat vestibular epithelium, using a rabbit antisynapsin I anti-serum. The staining was performed by immunofluorescence or by a peroxidase-antiperoxidase (PAP) technique. A strong immunoreactivity was observed with both methods. This immunoreactivity appeared as spherical patches distributed in the lower part of the epithelium. This distribution pattern is very similar to that of the efferent synaptic endings which form axodendritic synapses with the afferent nerve chalice of type I hair cells, or axosomatic synapses with type II hair cells. Some of the nerve chalices were also labelled; in this case, the immunoreactivity was more evident with PAP staining. These results thus suggest the presence of large amounts of synapsin I in the vestibular efferent nerve endings. These endings are known to be filled with numerous synaptic vesicles. This localization of synapsin I is well correlated with previous work that report a close association between synapsin I and small synaptic vesicles. The presence of synapsin I in sensory endings such as the afferent nerve chalices was unexpected and is under investigation.

Light- and electron microscopy of isolated vestibular hair cells from the guinea pig.

Cells isolated from the guinea-pig vestibular sensory epithelia were studied using light- and electron-microscopic techniques. The cells maintained their characteristic shapes when they had been separated. Mammalian vestibular cells are traditionally divided into two classes, type-I and type-II hair cells. It was, however, found that the population of isolated cells consisted of hair cells with a striking variability in shape and size. This was most conspicuous for the type-I hair cells. Isolated hair cells processed for electron microscopy showed that the isolation process caused minor ultrastructural damage but that the separation often was incomplete in that the large calyx-like nerve endings were still attached to type-I cells. The results suggest that the distinction of only two classes might be insufficient to describe mammalian vestibular hair cells.

Immunocytochemical localization of the GTP-binding protein G0 alpha in the vestibular epithelium and ganglion of the guinea-pig.

The guanine nucleotide binding protein G0 alpha was immunolocalized in the guinea-pig vestibular system by confocal and electron microscopy. The vestibular sensory epithelia consist of the macula utriculi, macula sacculi and cristae ampullaris of the semicircular canals. Two types of hair cells are present in these epithelia. Type I hair cells are surrounded by an afferent nerve calyx that receives efferent innervation and type II hair cells are innervated directly by the afferent and efferent nerves. G0 alpha protein was observed on the inner face of the afferent calyceal membrane surrounding type I hair cells and in nerve endings in contact with type II hair cells. No labelling was found in the stereocilia and cuticular plate of type I and type II hair cells whereas the cytoplasmic matrix displayed a diffuse labelling. The plasma membrane of the supporting cells showed discreet labelling in the confocal microscope that are still confirmed by electron microscopy. A positive reaction was also observed along the plasma membrane of the vestibular ganglion neurons. Immunoblotting with affinity-purified polyclonal rabbit antibodies selective for the 39 kDa alpha subunit of G0 indicated that G0 alpha protein was present in both the vestibular ganglion. That G0 alpha labelling was observed in the cytoplasm of vestibular hair cells and in nerve endings contacting hair cells suggests that G0 may be involved in the modulation of vestibular neurotransmission.

Changes in striatal cholinergic, gabaergic, dopaminergic and serotoninergic biochemical markers after kainic acid-induced thalamic lesions in the rat.

Kinetic parameters of 3H-choline, 3H-GABA and 3H-dopamine (DA) uptakes in striatal homogenates containing nerve endings were determined 2 to 3 weeks after kainic acid injection into the ipsilateral "centre médian"-parafascicular complex area of the thalamus in the rat. Results showed a marked decrease in 3H-choline uptake concomitant with a selective decrease in Vmax. Data also showed a large decrease in 3H-GABA uptake resulting from a decreased affinity of uptake sites for their substrate. These data were associated with the previously described decrease in choline acetyltransferase and increase in glutamic acid decarboxylase apparent activity, respectively. An apparent marked increase in 3H-DA uptake was likewise measured, mainly related to an increase in Vmax. Determination of serotonin (5HT) and 5-hydroxyindole acetic acid (5HIAA) endogenous contents showed in the deafferented striatum a decrease in 5HT concentrations associated with an increase in 5HIAA levels. Taken together, all these changes in neurotransmitter markers suggest that, directly through the thalamostriatal pathway or indirectly, the thalamus can exert a complex influence on striatal cholinergic and GABAergic neuronal functions as well as on the activity of dopaminergic and serotoninergic striatal afferent fibers.

Secretory function of the vestibular nerve calyx suggested by presence of vesicles, synapsin I, and synaptophysin.

Type I sensory hair cells of the vestibular epithelium are nearly completely ensheathed by an afferent nerve ending, the vestibular nerve calyx. We have recently reported that the nerve calyx, and, in particular, its apical portions surrounding the neck of the hair cell, are immunoreactive for synapsin I (Favre et al., 1986), a major membrane component of small synaptic vesicles of axonal endings. We have now found, by electron microscopy, that the same region of the calyx is densely populated by microvesicles morphologically similar to typical presynaptic small synaptic vesicles. Furthermore, we have established by light microscopy immunocytochemistry that this region of the calyx also contains a high concentration of synaptophysin, another well-characterized major component of small synaptic vesicle membranes. These results suggest that the upper portion of the calyx is equipped with the machinery that in presynaptic terminals is involved in the release of neurotransmitters and raise the possibility that the calyx, via secretion of neurotransmitterlike substances, might modulate the function of type I hair cells.

Pressure-induced basilar membrane position shifts and the stimulus-evoked potentials in the low-frequency region of the guinea pig cochlea.

We have used the guinea pig isolated temporal bone preparation to investigate changes in the non-linear properties of the tone-evoked cochlear potentials during reversible step displacements of the basilar membrane towards either the scala tympani or the scala vestibuli. The position shifts were produced by changing the hydrostatic pressure in the scala tympani. The pressures involved were calculated from measurements of the fluid flow through the system, and the cochlear DC impedance calculated (1.5 x 10(11) kg m-4 s-1, n = 10). Confocal microscopic visualization of the organ of Corti showed that pressure increases in the scala tympani caused alterations of the position of the reticular lamina and stereocilia bundles. For low pressures, there was a sigmoidal relation between the DC pressure applied to the scala tympani (and thus the position shift of the organ of Corti) and the amplitude of the summating potential. The cochlear microphonic potential also showed a pronounced dependence on the applied pressure: pressure changes altered the amplitude of the fundamental as well as its harmonics. In addition, the sound pressure level at which the responses began to saturate was increased, implying a transition towards a linear behaviour. An increase of the phase lag of the cochlear microphonic potential was seen when the basilar membrane was shifted towards the scala vestibuli. We have also measured the intracochlear DC pressure using piezoresistive pressure transducers. The results are discussed in terms of changes in the non-linear properties of cochlear transduction. In addition, the implications of these results for the pathophysiology and diagnosis of Meniérè's disease are discussed.

Perilymphatic fluid compartments and intercellular spaces of the inner ear and the organ of Corti.

An in vitro preparation of the inner ear cochlea has been used to visualize the structural relationships of unfixed, living sensory cells and structural components within the intact hearing organ. By perfusing perilymphatic compartments of the cochlea with fluorochrome-conjugated dextran, the extracellular spaces were clearly outlined. The staining pattern illustrated the large fluid compartments formed by the tunnel of Corti, the space of Nuel, and the outer tunnel. The dextran solution also indicated the spaces between the outer hair cell rows, the inner hair cells, and the surrounding supporting cells. The staining pattern demonstrates that the organ of Corti has a loose structure, suggesting a weak mechanical coupling between the cells. Moreover, it is evident that substances applied to the perilymph (e.g., therapeutic drugs) will readily reach all the cells of the hearing organ. In addition to the intraorgan fluid compartments, the spiral limbus was shown to contain significant volumes of perilymph within the intercellular spaces forming the so-called teeth of Huschke between the interdental cells. An extensive system of bundles following the teeth of Huschke was shown to be completely immersed in perilymph. The bundles were stained by a potentiometric dye, which in the inner ear primarily stains nerve fibers and sensory cells, which may indicate a nervous control of cells in this region.