| 1. |
- Flock, Å., et al.
(författare)
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Methods for integrating fluorimetry in the study of hearing organ structure and function
- 1997
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Ingår i: Hearing Research. - : Elsevier. - 0378-5955 .- 1878-5891. ; 106:1-2, s. 29-38
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Flock, Å., et al.
(författare)
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Supporting cells contribute to control of hearing sensitivity
- 1999
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Ingår i: Journal of Neuroscience. - : Society for Neuroscience. - 0270-6474 .- 1529-2401. ; 19:11, s. 4498-4507
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Fridberger, Anders, 1966-, et al.
(författare)
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Acoustic overstimulation increases outer hair cell Ca2+ concentrations and causes dynamic contractions of the hearing organ
- 1998
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 95:12, s. 7127-7132
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Fridberger, Anders, 1966-, et al.
(författare)
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Acute mechanical overstimulation of isolated outer hair cells causes changes in intracellular calcium levels without shape changes
- 1996
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Ingår i: Acta Oto-Laryngologica. - : Informa Healthcare. - 0001-6489 .- 1651-2251. ; 116:1, s. 17-24
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Tidskriftsartikel (refereegranskat)abstract
- Impaired auditory function following acoustic overstimulation, or noise, is mainly reported to be accompanied by cellular changes such as damage to the sensory hair bundles, but changes in the cell bodies of the outer hair cells have also been described. To investigate more closely the immediate cellular responses to overstimulation, isolated guinea pig outer hair cells were subjected to a 200 Hz oscillating water jet producing intense mechanical stimulation. The water jet was aimed at the cell body of the isolated outer hair cell. Cell shape changes were studied using video microscopy, and intracellular calcium concentration changes were monitored by means of the fluorescent calcium indicator Fluo-3. Cells exposed to a high-intensity stimulus showed surprisingly small light-microscopical alterations. The cytoplasmic calcium concentration increased in most cells, although some cells appeared very resistant to the mechanical stress. No correlation could be found be tween the calcium concentration changes and the cell length. The changes in calcium concentration reported here are suggested to be involved in the long-term pathogenesis of noise-induced hair cell damage.
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| 5. |
- Fridberger, Anders, 1966-, et al.
(författare)
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An in vitro model for acoustic overstimulation
- 1998
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Ingår i: Acta Oto-Laryngologica. - : Informa Healthcare. - 0001-6489 .- 1651-2251. ; 118:3, s. 352-361
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Tidskriftsartikel (refereegranskat)abstract
- Although many studies have been performed on the effects of acoustic overstimulation on the inner ear, our knowledge about the cellular processes underlying reduced hearing sensitivity and auditory cell death is still limited. In order to further our understanding of cellular processes occurring in conjunction with acoustic trauma, we designed an in vitro model to study the effects of overstimulation directly on sensory hair cells isolated from the low-frequency part of the guinea pig cochlea. The isolated outer hair cells were subjected to pressure jets delivered by a glass micropipette positioned close to the cell, in order to mimic the pressure changes occurring in the intact inner ear during sound stimulation. A second micropipette coupled to a piezoresistive pressure transducer was used as a probe measuring the pressure at precise locations at and around the cell. In a previous study, we found that such stimulation gave rise to increases in the intracellular calcium concentration. The present study characterizes the stimulus, describes the computer-controlled setup used for calibration, and gives examples of different modes of overstimulation at the cellular level. The peak pressure that could be generated using the pressure jet was around 325 Pa, or 144 dB (re 20 microPa) at 140 Hz. The pressure jet elicited large mechanical vibrations of the cell bodies of isolated cells. The vibration mode of the cells often changed over time, implying that the stimulation caused changes of the cellular stiffness. However, most cells appeared quite resistant to the high intensity mechanical stimulation.
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| 6. |
- Fridberger, Anders
(författare)
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Hair cell and organ of corti responses to normal and intense acoustic stimulation
- 1997
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The principal aims of the studies described in this thesis were to develop an in vitro model for studying acoustic overstimulation at the cellular level, to define the electrical and mechanical response characteristics of the perfused temporal bone preparation, and to investigate the effects of intense sound stimulation on the calcium levels of the hair cells in the intact hearing organ. In the in vitro model for acoustic overstimulation, isolated cochlear outer hair cells were subjected to a pressure jet emanating from a glass micropipette aimed at the cell body. The pressure jet was generated by the vibrating shaft of a minishaker, hydraulically coupled to the micropipette. Such stimulation was found to cause increases of the cytoplasmic calcium concentration in most auditory sensory cells. The calcium changes were sustained, and no evidence of recovery of the elevated levels were seen after the termination of the stimulus. A system was also developed to measure the pressures delivered to the cells. This system was based on a piezoresistive pressure transducer connected to a glass micropipette brought into the immediate vicinity of cells subjected to the pressure jet, allowing highly localized pressure changes to be measured. The peak pressure that could be generated by the stimulus system was found to be 144 dB SPL. Taking the middle ear transfer function into account, this level would correspond to approximately 120 dB SPL at the tympanic membrane during normal sound stimulation. Using laser heterodyne interferometry, the sound-induced vibrations of the low-frequency regions of the inner ear were investigated. The responses of the perfused isolated temporal bone preparation was found to be similar to that of living animals, both in terms of sharpness of tuning and the presence of nonlinearities. These characteristics were also reflected in the extracellularly recorded receptor potentials of the hair cells. The mechanical and electrical responses of the low-frequency regions of the cochlea were substantially different from the high-frequency regions, however. Methods were developed to load the organ of Corti with fluorescent dyes and to measure the fluorescence after various experimental manipulations, using video-enhanced microscopy. The fluorescence images were further processed off-line, using a computerized algorithm, to remove out-of-focus information. When the isolated temporal bone preparation was subjected to acoustic overstimulation, large increases of the calcium concentration of the outer hair cells were seen. In addition, overstimulation caused contractions of the hearing organ that were reversible after the termination of the stimulus. Both the calcium changes and the contraction response could be expected to have severe effects on the function of the inner ear.
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| 7. |
- Fridberger, Anders, 1966-, et al.
(författare)
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Pressure-induced basilar membrane position shifts and the stimulus-evoked potentials in the low-frequency region of the guinea pig cochlea
- 1997
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Ingår i: Acta Physiologica Scandinavica. - 0001-6772 .- 1365-201X. ; 161:2, s. 239-252
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Ulfendahl, M., et al.
(författare)
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Mechanical response characteristics of the hearing organ in the low-frequency regions of the cochlea
- 1996
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Ingår i: Journal of Neurophysiology. - : American Physiological Society. - 0022-3077 .- 1522-1598. ; 76:6, s. 3850-3862
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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