Genetic structure and diversity analysis of the primary gene pool of chickpea using SSR markers.

Members of the primary gene pool of the chickpea, including 38 accessions of Cicer arietinum, six of C. reticulatum and four of C. echinospermum grown in India were investigated using 100 SSR markers to analyze their genetic structure, diversity and relationships. We found considerable diversity, with a mean of 4.8 alleles per locus (ranging from 2 to 11); polymorphic information content ranged from 0.040 to 0.803, with a mean of 0.536. Most of the diversity was confined to the wild species, which had higher values of polymorphic information content, gene diversity and heterozygosity than the cultivated species, suggesting a narrow genetic base for cultivated chickpea. An unrooted neighbor-joining tree, principal coordinate analysis and population structure analysis revealed differentiation between the cultivated accessions and the wild species; three cultivated accessions were in an intermediate position, demonstrating introgression within the cultivated group. Better understanding of the structure, diversity and relationships within and among the members of this primary gene pool will contribute to more efficient identification, conservation and utilization of chickpea germplasm for allele mining, association genetics, mapping and cloning gene(s) and applied breeding to widen the genetic base of this cultivated species, for the development of elite lines with superior yield and improved adaptation to diverse environments.

Simultaneous measurements of ossicular velocity and intracochlear pressure leading to the cochlear input impedance in gerbil.

Recent measurements of three-dimensional stapes motion in gerbil indicated that the piston component of stapes motion was the primary contributor to intracochlear pressure. In order to make a detailed correlation between stapes piston motion and intracochlear pressure behind the stapes, simultaneous pressure and motion measurements were undertaken. We found that the scala vestibuli pressure followed the piston component of the stapes velocity with high fidelity, reinforcing our previous finding that the piston motion of the stapes was the main stimulus to the cochlea. The present data allowed us to calculate cochlear input impedance and power flow into the cochlea. Both the amplitude and phase of the impedance were quite flat with frequency from 3 kHz to at least 30 kHz, with a phase that was primarily resistive. With constant stimulus pressure in the ear canal the intracochlear pressure at the stapes has been previously shown to be approximately flat with frequency through a wide range, and coupling that result with the present findings indicates that the power that flows into the cochlea is quite flat from about 3 to 30 kHz. The observed wide-band intracochlear pressure and power flow are consistent with the wide-band audiogram of the gerbil.

The vibration pattern of the hearing organ in the waltzing guinea-pig measured using laser heterodyne interferometry.

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 tuned displacement response of the hearing organ is generated by the outer hair cells.

The motile responses of the guinea-pig hearing organ in response to a tone applied to the ear were measured by laser interferometry. Two types of responses can be recorded: (i) a vibration at the frequency of the applied tone; and (ii) a displacement response consisting of a shift in the position of the organ surface. The purpose of this study is to characterize the displacement response. The results are as follows. There is a relationship between the frequency of highest sensitivity (best-frequency) of the displacement response and the site from which it is recorded. High best-frequencies are noticed at more basal locations, low best-frequencies towards the apex. The displacement response is more frequency-selective than the vibration response. The displacement response is observed within physiological sound pressure levels. Its sharpness is dependent on the stimulus intensity, it shows biological variability and can be manipulated by drugs that are known to modify the receptor potential of the sensory cells, or to interfere with outer hair cell motility. These results suggest that the displacement response is an important step in the transduction process in the mammalian hearing organ and that it is generated by the motile action of the outer hair cells.

Mechanical demodulation of hydrodynamic stimuli performed by the lateral line organ.

Tonic displacements of the fish lateral line cupula were observed during stimulation of the organ with amplitude-modulated water motion. The modulation frequency was fixed at 2.4 Hz and the carrier frequency was varied from 25 to 500 Hz. The time waveforms of the cupular displacement at carrier frequencies below 280 Hz and above 470 Hz were essentially amplitude-modulated waves. Between 350 Hz and 410 Hz the magnitude at the modulation frequency increased sharply and the predominant shape of the displacement waveform changed to that of the modulating frequency. The mechanism for extraction of the modulation component may play a key role in the decoding of sensory information.

Shearing motion in the hearing organ measured by confocal laser heterodyne interferometry.

To investigate the presence of the postulated shearing motion in the micromechanics of the inner ear during sound stimulations we measured the vibratory response of the tectorial membrane and the reticular lamina in the third cochlear turn in an isolated temporal bone preparation using confocal laser heterodyne interferometry. The mechanical response of the tectorial membrane had the same frequency of maxima as the underlying reticular lamina, but was not as sharply tuned. When the two-dimensional motion was calculated from measurements made from several viewing angles it was found that the vibration of the reticular lamina had significant components both normal and tangential to its surface. The tectorial membrane motion, however, was primarily in a direction approximately perpendicular to the surface of the reticular lamina. The results indicate that shearing motion is produced predominantly by the radial motion of the reticular lamina.

Frequency-specific position shift in the guinea pig organ of Corti.

The organ of hearing is tuned as expressed both in the vibratory response of the cochlear partition and in the resulting receptor potentials of the sensory cells. We now demonstrate a sharply tuned response, consisting of a position shift of the surface of the organ of Corti, occurring during the presentation of a tone. The magnitude of the position shift exceeds that of the vibratory response to the stimulus. The shift is most pronounced in the region of the outer hair cells, and its affected by an inhibitor of outer hair cell motility. We conclude that the response is induced by the action of the outer hair cells.

Dose rate to the inner ear during Mössbauer experiments.

The most widely used technique for studying vibrations of the inner ear utilises the Mössbauer effect; this requires placement of a radioactive source on the basilar membrane. This source, although small in size and less than 37 MBq (1 mCi) in strength, is placed in close proximity to sensitive receptor cells. Using a series solution for the radiation field of a rectangular source the absorbed dose rate delivered to receptor cells at various depths and at points off-axis from the centre of the source is calculated. It is concluded that the dose delivered during the course of a Mössbauer experiment may well be sufficient to damage receptor cells and cause a loss of response.

Non-linear response to amplitude-modulated waves in the apical turn of the guinea pig cochlea.

Mechanical vibrations of the Hensen's cells were measured in the apical turn of the cochlea in living guinea pigs, in response to amplitude-modulated (AM) sound. The FFT of the input wave consisted of spectral components at the carrier frequency C and two sidebands (C+/-M) separated from the carrier by the modulation frequency M. The FFT of the velocity response consisted of components at: (i) the modulation frequency M, and harmonics n M; (ii) Carrier frequency C and sidebands (C+/-n M); (iii) harmonics of the carrier frequency and their side bands (2C+/-n M); (3C+/-n M); (4C+/-n M); em leader n=1,2,3, em leader,10. The carrier and the first pair of side bands were broadly tuned and nearly linear. Other components were sharply tuned and highly non-linear, suggesting a different origin. Evidence is presented that these components are generated in the non-linear stereocilia dynamics. An important function of this non-linearity is to demodulate the AM wave to extract information contained in the modulation.

Homodyne interferometer for basilar membrane measurements.

Techniques available for measuring the mechanical response of the inner ear are compared. These include capacitive probe, Mössbauer and interferometric methods. The theory of a homodyne interferometer utilized for inner ear measurements is given. Experimental apparatus built to test the interferometer performance is described. Experimental results show that the measuring system can detect vibrations as low as 3 X 10(-12) cm. Its frequency response is flat within 1 dB from 0.1 to 40 kHz. It has a linear dynamic range of over 90 dB. Immunity of the interferometer to various disturbances is demonstrated.

The response of the apical turn of cochlea modeled with a tuned amplifier with negative feedback.

In an earlier study [Hear. Res. 149 (2000) 55] velocity amplitudes of the outer Hensen's cell (HC) and basilar membrane (BM) were measured before, and at different times, after, sacrificing the animal. The velocity amplitude changed in a way that was characteristic of a negative feedback amplifier. A simple negative feedback amplifier model was proposed to explain the magnitude of the HC and BM velocity changes at CF. In the experiment tuning changed as well, both at the HC and BM. The model has now been extended to include tuning changes. The model response is compared with the experimental observations. The model is able to account quantitatively for the following experimental observations: (i) At the HC the tuning broadens and velocity decreases slowly after sacrifice. (ii) At the BM tuning sharpens and velocity increases at a faster rate. (iii) The velocity increase at BM is much larger than the decrease at HC.

Reticular lamina vibrations in the apical turn of a living guinea pig cochlea.

The reticular lamina of the apical turn of a living guinea pig cochlea was viewed through the intact Reissner's membrane using a slit confocal microscope. Vibrations were measured at selected identified locations with a confocal heterodyne interferometer, in response to tones applied with an acoustic transducer coupled to the ear canal. The position coordinates of each location were recorded. Mechanical tuning curves were measured along a radial track at Hensen's cells, outer hair cells, inner hair cells and at the osseous spiral lamina, over a frequency range of 3 kHz, using five sound pressure levels (100, 90, 80, 70 and 60 dB SPL). The carrier to noise ratio obtained throughout the experiments was high. The response shape at any measuring location was not found to change appreciably with signal level. The response shape also did not change significantly with the radial position on the reticular lamina. However, the response magnitude increased progressively from the inner hair cell to the Hensen's cell. The observed linearity of response at the fundamental frequency is explained by the presence of negative feed back in the apical turn of the cochlea.

Distribution of cochlear damage caused by the removal of the round window membrane.

The distribution of damage that occurs in the cochlea after removal of the round window membrane was examined in the apical, middle and basal regions with light and electron microscopy. The damage resembles that seen after acoustic trauma in many respects. The outer hair cells are often disrupted in damaged zones, and the radial afferent fibers to the inner hair cells swell enormously to form large vacuoles. 16 h after opening of round window, there is conspicuous swelling of myelinated axons in the osseous spiral lamina of the apical region. This swelling is associated with large vacuoles underneath the inner hair cells. 10 h after opening the round window, much smaller vacuoles are seen in the apical region. The distribution of the damage is not uniform throughout the cochlea. Damage is usually less severe and is not uniform in the middle region but is pronounced in the base. The nature of the damage is also variable in different animals. For example, sharply delimited, discontinuous damage to the inner hair cells was occasionally observed in the apical region. The most likely cause for the damage to the cochlea is a pressure differential across the organ of Corti that appears after removing the round window membrane. The damage apparently causes low frequency random movements of the basilar membrane that are observed in the experimental cochleas using a reflected laser beam.

Ultrastructural damage in cochleas used for studies of basilar membrane mechanics.

Cat cochleas used for interferometric studies of basilar membrane mechanics were examined with the electron microscope. The structures most severely damaged in the experimental cochleas are the outer hair cells and the radial afferent fibers to the inner hair cells. Since the basilar membrane and other supporting structures appear to be normal, mechanical changes observed in the experimental cochleas are most probably due to outer hair cell damage. Individual animals with varying degrees of damage showed large differences in the frequency of basilar membrane resonance at the same place in the cochlea. Shifts in tuning of this magnitude could occur as a consequence of hair cell damage only if the stiffness of the stereocilia and associated structures was greater initially than the stiffness of the basilar membrane and gradually decreased with damage. The present series of observations, therefore, suggest that the stiffness of the outer hair cell stereocilia determines basilar membrane tuning.

Modelling the malleus vibration as a rigid body motion with one rotational and one translational degree of freedom.

Vibration of a set of points distributed along the manubrium of cat was measured with a heterodyne interferometer in response to sinusoidal acoustic signals. The observed motion did not fit pure rotation of the malleus around a fixed axis coinciding with the anterior mallar and posterior incudal ligament as is classically assumed. As a first approximation a model of motion consisting of a rotational and a translational component was used. At low frequencies the rotation is mostly predominant, but the situation may be entirely reversed at mid and high frequencies. The presence of a translation besides rotation was also found at some frequencies in the motion of the human malleus.

Spectral characteristics of the responses of primary auditory-nerve fibers to amplitude-modulated signals.

The spectral responses of cat single primary auditory nerve fibers to sinusoidal amplitude-modulated (AM) and double-sideband (DSB) acoustic signals applied to the ear were examined. DSB is an amplitude-modulated signal with a suppressed carrier. Period histograms were compiled from the neural spike-train data, and the frequency spectrum was determined by Fourier transforming these histograms. For DSB signals, spectral components were found to be present at the frequencies of the stimulus as well as at certain combination frequencies. For AM signals, several clusters of spectral components were present. The lowest-frequency cluster consisted of components at DC, at the modulation frequency, and at its harmonics. A higher frequency cluster occurs around a component with the frequency of the carrier. The components of cluster are separated from the carrier by the modulation frequency and its harmonics. Yet higher-frequency clusters appear around multiples of the carrier frequency with components at frequencies separated from these multiples by the modulation frequency and its harmonics. The magnitudes of these spectral components were determined for carrier frequencies located below, at, and above the characteristic frequency of the units, and for different stimulus levels, modulation frequencies, and modulation depths. The low-frequency components present in the neural spike train appear to be the result of demodulation taking place in the inner ear. The demodulated components are strong and are present over a wide range of sound levels, carrier frequencies, modulation frequencies, and nerve-fiber characteristics. This demodulation may be significant for speech recognition.

Spectral characteristics of the responses of primary auditory-nerve fibers to frequency-modulated signals.

The spectral responses of cat single primary auditory nerve fibers to sinusoidal frequency-modulated acoustic signals applied to the ear are examined. Period histograms were constructed from the neural spike-train data, and the frequency spectrum was determined by Fourier transforming these histograms. Several clusters of spectral components were present. The lowest-frequency cluster consists of components at DC, at the modulation frequency, and at its harmonics. In the next cluster, components surround the carrier frequency and are separated from it by the modulation frequency and its harmonics. Higher-frequency clusters surround frequencies that are twice and three times the carrier frequency. The components in each cluster are separated from the multiples of the carrier frequency by the modulation frequency and its harmonics. The magnitudes of the spectral components were investigated for carrier frequencies located below, at, and above the unit characteristic frequency, and for different signal levels, modulation frequencies, and modulation indices. The components at the modulation frequency and its harmonics were strong and present over a wide range of signal levels, carrier frequencies, modulation frequencies, and nerve-fiber characteristics. The presence of components at the modulation frequency indicates that a demodulation process is occurring. This process may be significant for speech recognition.

Measurement of basilar membrane vibrations and evaluation of the cochlear condition.

The experimental procedure for measuring basilar membrane responses to acoustic signals is described. The surgical procedure developed for opening the cochlea with minimal trauma is presented. Each experiment included sound pressure level measurements to define the input signal, cochlear microphonic (CM) measurements to monitor the cochlear condition, interferometric measurements and histological evaluation of the cochleas. The characteristic frequency (CF) and the sensitivity at CF for the basilar membrane response is correlated with the change of CM response observed in six animals. It is demonstrated that both tuning characteristics are extremely sensitive to cochlear trauma as evidenced by changes of CM.