View OC Motion
The cross section of an inner ear is shown. The entire preparation is vibrated while sitting on a holder that is attached to a piezo actuator. Thus, the mechanical vibration is transmitted via the bone to the inner ear structures. In the example shown, the preparation is vibrated at 1200 Hz. During one stimulus cycle 18 video images of the cross section were captured while strobe illumination was applied. For the animation the 18 frames that were captured are shown at a much slower rate, such that the movements of the inner ear structures can be seen by visual observation. Note that the movements of the basilar membrane result in a shear movement between tectorial membrane and reticular lamina.

View Animation
The cross section of a middle cochlear turn is shown. A sealed glass pipette that is mounted to a piezo bender serves as a paddle, and is placed below the basilar membrane into scala tympani. In the example shown, the paddle is vibrated at 1400 Hz. The vibrations are coupled via the fluids to the basilar membrane. During one stimulus cycle 18 video images of the cross section were captured while strobe illumination was applied. Off line, the velocity of s elected points is calculated between consecutive images using the Lukas-Kanade algorithm. The resulting Lissajous figures give the 80 times magnified displacement between the images. The up and down movement of the basilar membrane results in a radial movement of the reticular lamina.

View IHC Motion
The highly magnified view of the cross section of an inner ear is shown (basal turn). A sealed glass pipette that is mounted to a piezo bender serves as a paddle, and is immersed below the basilar membrane into scala tympani. In the example shown, the paddle is vibrated at 6000 Hz. The vibrations are coupled via the fluids to the basilar membrane. During one stimulus cycle 18 video images of the cross section were captured while strobed illumination was applied. For the animation the 18 frames that were captured are shown at a much slower rate, such that the movements of the inner ear structures (inner hair cell stereocilia bundle and Hensen's stripe of the tectorial membrane) can be seen by visual observation. Note that the radial vibration of the tectorial membrane is minimal. During one stimulus cycle the inner hair cell stereocilia bundle pivots. Further, the tip of the stereocilia bundle moves relative to the Hensen's stripe indicating no tight connection between the stereocilia bundle and the tectorial membrane.

Summary

NSF funding was available between 2000 and 2004 (IBN-0077476) and is currently available 2004-2007 (IBN-00415901) for a project entitled "Micromechanics of the mammalian cochlea".  Significant research progress has been made during the funding period.  The research results will be summarized as follows: (1.1.) anatomical studies in four different strains of mice, (1.2.) improvement of the video flow technique, (1.3.) driving point stiffness measurements made in vivo and in the hemicochlea to validate use of the hemicochlea for stiffness measurements, (1.4.) stiffness measurements in developing animals to examine the mapping of the frequency place code in the basal gerbil cochlea, (1.5.) a collaboration wherein compound action potential tuning curves were measured in developing gerbils, (1.6.) stiffness measurements on isolated pillar cells, (1.7.) the hemicochlea as a tool to evaluate potential distributions in the electrically stimulated cochlea, (1.8.) motion analysis in the hemicochlea, (1.9.) tectorial membrane, reticular lamina, and organ of Corti stiffness measurements, and (1.10.) tectorial membrane, reticular lamina and inner hair cell stereocilia bundle kinematics.  In addition to furthering understanding of normal cochlear function, this research has resulted in the training of students, the development of teaching material, and the preparation of instructional materials for lay audiences.  That broad impact will be summarized under (1.11.).  Furthermore, a concerted effort was made to involve high-school teachers in this research.  A high school teacher was hired for the research during summer 2004.  Throughout the following school year, the PI provided an enrichment class to the students of Foreman High-School in Chicago. 

1.1 Cochlear dimensions obtained in hemicochleae of four different strains of mice: CBA/CaJ, 129/CD1, 129/SvEv and C57BL/6J. Because homologies between mice and human genomes are well established and hereditary abnormalities are similar, mice present a valuable animal model for studying hereditary hearing disorders in humans.  One of the manifestations of hereditary hearing disorders might lie in the structure of the cochlea, for example, in changes of gross morphology.  Cochlear dimensions are one of the factors that determine inner ear mechanics and resulting hearing function.  Therefore, gross cochlear dimensions might underlie hearing differences in different strains of mice.  Although several studies have examined mouse inner ear structures on a sub-cellular level, only a few have studied cochlear gross morphology.  Moreover, the sparse available data were acquired from fixed and dehydrated tissue.  Dehydration produces severe distortion of gel-like cochlear structures such as the tectorial membrane (TM) and the basilar membrane hyaline matrix (Edge et al., 1998).  In contrast, our hemicochlea technique, which allows fresh mouse cochlear material to be viewed from a radial perspective, was used to evaluate the dimensions of gross cochlear structures in four mouse strains: CBA/CaJ, 129/SvEv, 129/CD1 and C57BL/6J.  Except for the CBA/CaJ, these strains are known to possess genes for age-related hearing loss.  The measurements showed no major differences among the four strains.  However, when compared with data in the literature, thickness measures of the basilar membrane were up to 10 times larger.  Such differences are likely to result from the different techniques used to process the material.  The hemicochlea technique eliminates much of the distortion and provides a closer approximation of in vivo conditions.  These data have been published (Keiler and Richter, 2001).

1.2. Motion analysis in the hemicochlea. Movements of selected cochlear structures, which were captured in a sequence of images, can be retrieved.  In a first step, we showed that local brightness is conserved.  The resulting system then can be solved either by using an Eulerian or a Lagrangian description of the conservation equations.  In the first approach, a first-order linear partial differential equation for a stream function is solved.  Streamlines are constant values of the stream function, and velocity vectors are tangent to the streamlines.  The integration of the ratio of temporal to spatial intensity gradients along characteristic curves, which are isointensity contours, determines the stream function.  In the second approach, the displacement of isointensity contours on sequential images determines the normal component of velocity of an area element, while the tangential component is computed from the local constant area constraint.  We have validated our methods using pairs of images generated from our calculations of the vibrational deformation in a cross section of the organ of Corti in the mammalian cochlea, and applied the Lagrangian method to images obtained from the hemicochlea preparation.  The results have been published (Cai et al., 2002; Cai et al., 2003).

1.3. In vivo and in vitro stiffness measurements in the mammalian cochlea. Basilar membrane stiffness measurements were made in two preparations using custom-built piezoelectric sensors.  The first in vivo preparation allowed access to the tissue at a location approximately 2.5 mm from the basal end of the basilar membrane.  The second in vitro hemicochlea preparation allowed access to the tissue at multiple locations spanning the entire cochlear length.  Stiffness data from the hemicochlea were verified by direct comparison with the data obtained in vivo.  Results have been published (Emadi and Dallos, 2000; Emadi et al., 2002a; Emadi et al., 2002b; Emadi et al., 1999; Emadi et al., 2004).

1.4. Developmental changes of mechanics in the gerbil cochlea. This study was the first experimental examination of basilar membrane stiffness during the development of cochlear function.  It has been established previously that the most effective sound frequencies (best frequencies) for some locations in the gerbil cochlea change during the maturation of hearing (e.g. Harris and Dallos, 1984 Aug 17).  Changes of tissue stiffness have been postulated to contribute to this shift of best frequencies.  Structures in a hemicochlea preparation were stimulated at audio frequencies using a glass paddle and best frequencies of motions were determined with a computer-based imaging system.  In separate experiments, a piezoelectric-based sensor was used to measure the point stiffness of the basilar membrane.  Both types of measurements were taken at multiple ages spanning the onset and maturation of hearing.  Confirming the results from previous studies, the motion data demonstrated a developmental shift of best frequency in the basal turn but no such shifts in the middle turn.  The stiffness data revealed a developmental change of stiffness in the basal turn but no statistically significant shift in the middle turn after the onset of cochlear function.  A spring-mass resonance model that quantitatively combines the measured stiffness changes with estimated mass changes showed that stiffness is likely to be an important factor in determining best frequency after the onset of hearing in the gerbil.  Results have been presented as posters (Emadi et al., 2001; Richter et al., 2000) and a paper is in final preparation (Emadi and Richter, 2005).

1.5. High-frequency sensitivity of the mature gerbil cochlea and its development. Thresholds of compound action potentials evoked by tone pips were measured in cochleae of anesthetized gerbils, both in adults and in neonates aged 14, 16, 18, 20 and 30 days, using round-window electrodes.  Stapes vibrations also were measured, using a laser velocimeter, in many of the same animals.  The stapes measurements assessed intrinsic cochlear sensitivity in isolation from middle ear effects, and circumvented problems associated with calibration of acoustic stimuli at high frequencies.  Whether referenced to sound pressure level in the ear canal or stapes vibration velocity, thresholds in adults were roughly uniform in the entire range of frequencies tested (1.25-38.5 kHz).  In neonates, thresholds decreased systematically as a function of age, with the largest reductions occurring at the highest frequencies.  Thirty days after birth, thresholds at all frequencies still had not reached the sensitivity of adult animals.  The results for adult gerbils are consistent with the recent finding that basilar-membrane responses to CF tones normalized to stapes vibrations are as sensitive at sites near the round window as at more apical locations (Overstreet and Ruggero, 2002; Overstreet et al., 2002a; Overstreet et al., 2002b).  The results for neonates confirm that the extreme basal region of the cochlea is the last to develop, with substantial changes occurring between 20 and 30 days after birth.  A paper has been published (Overstreet et al., 2003).

1.6. Effects of ATP on the stiffness of mammalian inner ear structures. Inner and outer pillar cells form the arch of Corti, separating the areas of inner and outer hair cells.  This arch is a rigid structure influencing the micromechanics of the organ of Corti.  Rigidity is conferred to these cells by bundles of parallel microtubules and actin filaments packed in long slender rods. The stiffness of the microtubular bundles depends on the degree of the cross-linking of the filaments, and damage to connections between parallel microtubules decreases the stiffness of pillar cells (Tolomeo and Holley, 1997).  Physiologically, microtubule-associated proteins (MAPs) control these cross-links, and MAPs, in turn, are regulated by calcium-dependent phosphorylation.  Since intracellular calcium is modulated by adenosine triphosphate (ATP) in pillar cells of the guinea pig (Chung and Schacht, 2001), we hypothesized that cell stiffness may be controlled by ATP.  This hypothesis was tested in isolated gerbil and guinea pig pillar cells.  The bending stiffness for cells of different lengths was measured with stiffness-calibrated glass fibers.  Furthermore, the response of intracellular calcium levels to the application of extracellular ATP was determined using fluo-3, a fluorescent calcium indicator that increases its fluorescent emission upon binding to nanomolar concentrations of free calcium.  Application of ATP to the experimental bath significantly increased intracellular calcium concentration of isolated guinea pig pillar cells.  Moreover, application of ATP into the experimental bath significantly changed the bending stiffness of the cell.  Stiffness changes were observed in 3 out of a total of 4 cells.  In contrast to guinea pigs, in gerbils application of ATP to the experimental bath did not significantly change the intracellular calcium concentration in 20 cells.  Bending stiffness of isolated gerbil pillar cells did not change significantly.  Results have been presented as a poster (Richter et al., 2002) and a paper is in preparation.

1.7. The hemicochlea as a tool to evaluate potential distributions in the electrically stimulated cochlea. The design and putative efficacy of cochlear-implant electrodes are based primarily upon indirect measurements, theoretical assumptions, and predictions from cochlear models.  Few real measurements of the potential distributions resulting from electrical stimulation have been made in a real inner ear because of the difficulty in accessing an intact cochlea.  Experiments compared electrical properties (longitudinal resistance, length constants) in an intact cochlea and in open and sealed hemicochleae to determine the conditions under which the hemicochlea can serve as a model of the electrically stimulated intact cochlea.  Data show that the sealed hemicochlea provide a reasonable model for determining potential distributions within the cochlea in a radial plane and for identifying the cochlear structures targeted by a cochlear-prosthesis electrode.  In addition, potential fields generated by a pair of electrodes placed in a saline bath were compared to results from the same electrodes placed in the gerbil hemicochlea in a monopolar or bipolar configuration.  Comparisons indicate the substantive effects of cochlear electroanatomy on the location of maximum potential difference across the organ of Corti and spiral ganglion cells.  Results have been presented as posters (Richter and Koch, 2002; Richter et al., 2001a; Richter et al., 2001b; Richter et al., 2001c).

1.8. Basilar membrane and organ of Corti micromechanics in the hemicochlea. Vibration patterns seen in a cross-section of a hemi-turn in a hemicochlea show how cochlear structures move when the active processes are absent.  In order to study vibration patterns of different structures of the inner ear and to test for multiple modes of vibration in a radial cochlear cross section (Kuile, 1900), two types of experiments were performed in gerbil hemicochleae.  In the first series of experiments, the entire cochlear preparation was vibrated and allowed to simultaneously stimulate all elements of the cochlear cross section attached to the bony scaffold.  Magnitude and phase differences were determined between adjacent structures.  In the second experimental series, a paddle in scala tympani was used to mechanically elicit basilar membrane vibrations and to study dominant modes of vibration that evince in a cochlear cross section.  Because both sets of experiments produced similar results, the data were combined.  The data show that (1) the basilar membrane moves up and down like a ribbon attached to two edges, the spiral limbus and the spiral ligament, and (2) the organ of Corti pivots around a fulcrum close to the foot of the inner pillar cell.  Vibrations of the organ of Corti can be separated into the movement of a “rotating wedge” and the movement of Hensen’s cells.  The rotating wedge consists of inner and outer pillar cells, inner and outer hair cells, Deiters’ cells and parts of the basilar membrane.  Results have been presented during workshops (Richter and Dallos, 2001; Richter and Dallos, 2003) and a paper has been submitted, has been reviewed and is currently revised (Richter and Dallos, 2005).

1.9. Stiffness as a function of location along the length of the cochlea. Stiffness of the tectorial membrane changed along the cochlea, with the TM being more compliant in the apex compared to the base. The tectorial membrane stiffness gradient (–3.0dB/mm) was smaller when compared to the basilar membrane stiffness gradient (–4.5dB/mm) obtained from the same animals. When the stiffness was measured above outer hair cell 1 close to the pillar cells, the stiffness was similar to the stiffness obtained at the basilar mid pectinate zone. Again, stiffness decreased from base to apex (-3.8dB/mm). In general, stiffness at the reticular lamina depended on the radial location and was larger above the first outer hair cell compared to the third outer hair cell.  Interestingly, tectorial membrane stiffness was similar to the stiffness of the reticular lamina above the second outer hair cell, was stiffer than the stiffness measured above the third row of outer hair cells and was more compliant than the stiffness above the first row of outer hair cells.  The results have been presented as posters (Emadi et al., 2002b) and two manuscripts are in final preparation (Richter et al., 2005b; Richter et al., 2005c).

1.10. Reticular lamina (RL) displacement. The reticular lamina was deflected with a rigid probe. Independent of the probes placement above outer hair cell 1 through 3, the reticular lamina moved like a rigid beam.  Reticular lamina movement couples to the movement of the inner hair cells.  While the reticular lamina is pushed down, the inner hair cell cuticular plate moves upwards towards the tectorial membrane.  A sea-saw like motion with the pivot point at the tip of the pillar cells can be observed.A stiffness gradient exists from the reticular lamina to the basilar membrane.  A stiff probe is used to deflect the reticular lamina. The displacement of the reticular lamina, the outer hair cell nuclei, the Deiters’ cell nuclei and the basilar membrane upper fiber band was measured.  The displacement of the reticular lamina was given by the displacement of the probe (steps of 5µm). The displacement of the outer hair cell nuclei was significantly smaller than the reticular lamina displacement.  The displacement of the Deiters’ cell nuclei was in the order of the displacement of the outer hair cell nuclei, and the displacement of the basilar membrane was not measurable.  The results indicate that a stiffness gradient of cochlear structures exist between the reticular lamina and the basilar membrane. While a compliant region exists across the outer hair cells, the stiffness seems to increase towards the basilar membrane.  The results are published (Richter and Quesnel, 2005).