In some pathological conditions excessive amounts of endolymph can accumulate and cause swelling, hydrops, of the membranous labyrinth. Reissner’s membrane in the cochlea will distend and may even rupture. We have studied the effects of acute hydrops, followed for up to 5–6 h, in a preparation that allows continuous monitoring of structural alterations in individual cells in Reissner’s membrane. This is accomplished by using laser confocal microscopy on the membrane visualized by labeling its cells with fluorescent dyes. In specimens subjected to hydrops it was observed that discrete structural defects developed in Reissner’s membrane. These were seen as lesions in single cells or in groups of cells in the epithelial layer. It is suggested that through these micro-lesions the electro-chemical environment of the organ of Corti can be altered causing hearing loss and tinnitus during hydrops.

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.

The Reissner's membrane (RM) was visualised by confocal microscopy in the isolated temporal bone of the guinea pig. The function of the organ was followed by measuring its physiological response. Static pressure applied in the basal coil caused a distention of the RM in the apical coil into the scala vestibuli. The sensitivity to a test tone was reduced. When the pressure was relieved, the RM returned to its original position and the response recovered. If the increased pressure was maintained, the RM would bulge further. The RM could then be reversibly stretched and return gradually, with a delay, to its original position. Alternatively, it could be over-stretched and return with an over-shoot past its original position toward the organ of Corti. In response to repetitive tone pulses of above 80 dB, hydrops of the RM also developed. This was accompanied by a reduced sensitivity. A slow recovery to the original position, or over-shoot, and return of responsiveness could be seen. Above 106 dB sustained loss was generally seen. It is concluded that the RM can accommodate increased scala media pressure by distention. This will relieve the organ of Corti from part of the pressure and may protect the organ from trauma.

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.

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.

Local anesthetics applied in the tympanic cavity have earlier been shown to affect the gross receptor potentials in reducing the cochlear microphonics and increasing the positive summating potential. To study the effects of this drug on the mechanical responses in the cochlea, vibrations were measured using laser heterodyne interferometry in an isolated in vitro temporal bone preparation from the guinea pig. Measurements were made at a set of frequencies in the fourth cochlear turn from the Hensen’s cells and the outer hair cells in response to sound applied to the ear. The tuning curves of the fundamental and the second harmonic components of the vibratory responses were plotted. When 2 mM tetracaine was applied, the high frequency slope of the second harmonic curve shifted down in frequency, this caused the frequency of the maximum of second harmonic tuning to shift down. These changes were reversible when tetracaine was washed out. Observations were also made in the temporal bone preparation in vitro with a confocal microscope. Fluorescent probes were used to label various structures in the organ of Corti. Optical sections were obtained by tilting the organ permitting a view from the side like a radial section through the organ. Images were acquired before, during and after application of tetracaine and were later analyzed with a computer program. Simultaneously, cochlear microphonics and the summating potential were obtained to monitor the electrical response of the preparation. Although the cochlear microphonics and summating potential decreased when 2 mM tetracaine was applied, structural changes were not measurable in the organ of Corti. The decrease was reversible when tetracaine was washed out. It is concluded that tetracaine affected the high frequency part of the non-linear second harmonic component, possibly by lowering the stiffness of the stereocilia bundle or the body of the outer hair cells.

Although initially met with scepticism, the in vitro temporal bone preparation of the cochlea has proved to be a very important tool for investigating the function of the mammalian auditory system. As present techniques are able to maintain sufficient cellular viability, the in vitro preparation offers a valuable bridge between investigations using isolated outer hair cells and the intact system in vivo.

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.

The dynamic responses of the hearing organ to acoustic overstimulation were investigated using the guinea pig isolated temporal bone preparation. The organ was loaded with the fluorescent Ca2+ indicator Fluo-3, and the cochlear electric responses to low-level tones were recorded through a microelectrode in the scala media. After overstimulation, the amplitude of the cochlear potentials decreased significantly. In some cases, rapid recovery was seen with the potentials returning to their initial amplitude. In 12 of 14 cases in which overstimulation gave a decrease in the cochlear responses, significant elevations of the cytoplasmic [Ca2+] in the outer hair cells were seen. [Ca2+] increases appeared immediately after terminating the overstimulation, with partial recovery taking place in the ensuing 30 min in some preparations. Such [Ca2+] changes were not seen in preparations that were stimulated at levels that did not cause an amplitude change in the cochlear potentials. The overstimulation also gave rise to a contraction, evident as a decrease of the width of the organ of Corti. The average contraction in 10 preparations was 9 microm (SE 2 microm). Partial or complete recovery was seen within 30-45 min after the overstimulation. The [Ca2+] changes and the contraction are likely to produce major functional alterations and consequently are suggested to be a factor contributing strongly to the loss of function seen after exposure to loud sounds.

We have used the perfused guinea-pig temporal-bone preparation to study the sound-evoked efflux of aspartate and glutamate, which are putative afferent transmitters in the cochlea. The cochlea was stimulated with white noise at 89, 95, and 101 dB SPL. Cochlear function was monitored by recording the endocochlear potential, the cochlear microphonic, and the summating potential. In silence, there was a low basal efflux of both amino acids. A significant and intensity dependent sound-evoked efflux of aspartate was observed at all levels, whereas a significant efflux of glutamate was found only at the 101 dB SPL level. Immunohistochemistry of sections from the organ of corti showed an ubiquitous distribution of glutamate-like immunoreactivity in the sensory organ and ganglion, whereas aspartate-like immunoreactivity was found in the region of the inner hair cells and in the spiral ganglion. In view of these findings, we suggest that not only glutamate, but also aspartate may have a neurotransmitter role in the afferent pathway of the cochlea.

The detection of sound by the mammalian hearing organ, the organ of Corti, is far from a passive process with the sensory cells acting as mere receptors. The high sensitivity and sharp tuning of the auditory apparatus are very much dependant on the active mechanical behavior of the outer hair cells, acting as effector cells.

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.

Local anaesthetics have been used intravenously and intratympanally to reduce tinnitus. In order to clarify its action in the periphery, we applied 0.5 mM tetracaine in the scala tympani in 18 cochleae and studied the effects on the receptor potentials. We used a temporal bone preparation of the guinea pig ear in vitro exposing the fourth cochlear turn where the cochlear microphonics (CM) and the summating potential (SP) were recorded. The perfusion was kept at a rate of 50 microliters/min. The frequency response of the cochlea was determined at the beginning of each experiment and the responses were recorded at the best frequency of the preparation. In another five cochleae an accumulated dose-response relationship was determined by increasing the tetracaine concentration in steps (50, 100, 300, 500, 1000 and 2000 microM), measuring the difference in amplitude of the receptor potentials. The CM decreased significantly (p < 0.001; mean 0.37 mV; SD 0.29). In 12 cochleae the SP was initially positive and did not increase significantly (p = 0.16; mean 0.07 mV; SD 0.16). In six cochleae the SP was initially negative and all changed polarity to positive and increased significantly (p < 0.05; mean 0.36 mV; SD 0.28). The effects on both the CM and the SP were reversible. Owing to the inter-individual variation between the cochleae the SP/CM ratio was determined and it increased significantly (p < 0.001; mean 0.18; SD 0.11). In the accumulated dose-response experiments the CM decreased significantly (p < 0.05) in a dose-dependent way, whereas the SP did not increase significantly. The SP/CM ratio increased significantly (p < 0.05) at 300 microM and 500 microM. We hypothesize that the peripheral tinnitus-reducing action of local anaesthetics is in part due to a reversal of the SP, but also to a reduction of the CM. The difference in effect of tetracaine on the receptor potentials, the CM and the SP, suggests that the SP is not dependent on the CM.

We have visualised nitric oxide (NO) released from the electrically stimulated myenteric plexus and hypogastric nerve. NO was visualised by a reaction with luminol and hydrogen peroxide to generate photons which were counted using a microscope coupled to a photon counting camera. Electrical stimulation of the tissues induced an increase in photon counts which was frequency-dependent and prevented by inhibition of the NO synthase or by tetrodotoxin. The light emitted during nerve stimulation was not only observed at the nerve terminals but also at the axon and soma. Our results indicate that NO released from the whole nerve cell may affect target cells surrounding all parts of the nitrergic neuron. Thus, NO functions as a unique mechanism of synaptic and non-synaptic communication in the nervous system. (C) 1997 Wiley-Liss, Inc.

Examination of the neuromasts in the tail of the living juvenile axolotl Ambystoma mexicanum with interference contrast (Nomarski) microscopy shows that their cellular structures can be identified in considerable detail. Microscopy was performed with a 40X water immersion objective or with a 100X objective in a tail insertion chamber. Thus the sensory hair bundles can be seen and their orientation can be determined. Large spheres in the basal part of the sensory cells were predicted to be synaptic bodies. Subsequent examination in the transmission electron microscope of the cells observed in the light microscope confirmed this notion. Inspection during periods of several hours reveals definite movements of the synaptic bodies.

The measurement of function in the intact organ of Corti has up to now been achieved by three methods: electrophysiology, mechanical measurement and biochemical analysis. The two former methods have supplied information at the level of single identified cells. We have used a fourth method, optical fluorimetry, to measure hair cell function at the cellular level in the intact organ of Corti. Here we describe the methods involved in fluorescence labelling and video-enhanced microscopy in combination with electrophysiological recording of cochlear microphonic (CM) and summating potentials (SP). The guinea pig temporal bone containing an intact ear drum, ossicular chain and cochlea can be maintained in the isolated state by perfusion of the scala tympani with oxygenated tissue culture medium. Substances added to the perfusate readily diffuse through the basilar membrane into the organ of Corti. In this way cells in the organ can be stained by a number of fluorescent probes which label different structures and functions. Here we have used two dyes which label mitochondria and fluoresce with an intensity proportional to metabolic activity. By simultaneous measurement of CM and SP the functional state of the organ can be monitored.

1. With the use of an in vitro preparation of the guinea pig temporal bone, in which the apical turns of the cochlea are exposed, the mechanical and electrical responses of the cochlea in the low-frequency regions were studied during sound stimulation. 2. The mechanical characteristics were investigated in the fourth and third turns of the cochlea with the use of laser heterodyne interferometry, which allows the vibratory responses of both sensory and supporting cells to be recorded. The electrical responses, which can be maintained for several hours, were recorded only in the most apical turn. 3. In the most apical turn, the frequency locations and shapes of the mechanical and electrical responses were very similar. 4. The shapes of the tuning curves and the spatial locations of the frequency maxima in the temporal bone preparation compared very favorably with published results from in vivo recordings of hair cell receptor potentials and sound-induced vibrations of the Reissner's membrane. 5. Compressive nonlinearities were present in both the mechanical and the electrical responses at moderate sound pressure levels. 6. The mechanical tuning changed along the length of the cochlea, the center frequencies in the fourth and third turns being approximately 280 and 570 Hz, respectively. 7. The mechanical responses of sensory and supporting cells were almost identical in shape but differed significantly in amplitude radially across the reticular lamina.

The mechanical tuning characteristics of the hearing organ were measured in response to sound stimulation using laser heterodyne interferometry in in vitro preparations of temporal bones from waltzing guinea-pigs expressing different degrees of hearing organ and sensory cell degeneration. Measurements were made at various stages of structural changes allowing us to correlate structure and mechanical function. It was found that the characteristic frequency of the response at a given location in the cochlea occurred at lower frequencies than what is normally seen and that the sharpness of the mechanical tuning was considerably reduced when sensory hair cells were absent and the hearing organ structurally altered. However, even when extensive hair cell degeneration was evident a residual mechanical tuning was present. These results further support the concept that the sensory hair cells plays a key role in determining normal auditory tuning characteristics. It is suggested that the basilar membrane mechanics gives rise to a broadly tuned mechanical response on which a sharper tuning mechanism, originating from the hair cells, is superimposed.

The continuous wavelet transform (CWT) and the short-time Fourier transform (STFT) were used to analyze the time course of cellular motion in the guinea pig inner ear. The velocity responses of individual outer hair cells and Hensen's cells to amplitude modulated (AM) acoustical signals applied to the ear canal displayed characteristics typical of nonlinear systems, such as the generation of spectral components at harmonics of the carrier frequency. Nonlinear effects were particularly pronounced at the highest stimulus levels, where half-harmonic (and sometimes quarter-harmonic) components were also seen. The generation of these components was consistent with the behavior of a dynamical system entering chaos via a period-doubling route. A negative-stiffness Duffing oscillator model yielded period-doubling behavior similar to that of the experimental data. We compared the effectiveness of the CWT and the STFT for analyzing the responses to AM stimuli. The CWT (calculated using a high-Q Morlet-wavelet basis) and the STFT were both useful for identifying the various spectral components present in the AM velocity response of the cell. The high-Q Morlet wavelet CWT was particularly effective in distinguishing the lowest frequency components present in the response, since its frequency resolution is appreciably better than the STFT at low frequencies. Octave-band-based CWTs (using low-Q Morlet, Meyer, and Daubechies 4-tap wavelets) were largely ineffective in analyzing these signals, inasmuch as the frequency spacing between neighboring spectral components was far less than one octave.