Enhanced contrast in X-ray microtomographic images of the membranous labyrinth using different X-ray sources and scanning modes.

The vestibular system, located in the inner ear, plays a crucial role in balance and gaze stabilisation by sensing head movements. The interconnected tubes with membranous walls of the vestibular system are located in the skull bone (the 'membranous labyrinth'). Unfortunately, these membranes are very hard to visualise using three-dimensional (3D) X-ray imaging techniques. This difficulty arises due to the embedment of the membranes in the dense skull bone, the thinness of the membranes, and the small difference in X-ray absorption between the membranes and the surrounding fluid. In this study, we compared the visualisation of very small specimens (lizard heads with vestibular systems smaller than 3 mm) by X-ray computed micro-tomography (µCT) based on synchrotron radiation and conventional sources. A visualisation protocol using conventional X-ray µCT would be very useful thanks to the ease of access and lower cost. Careful optimisation of the acquisition parameters enables detection of the membranes by using µCT scanners based on conventional microfocus sources, but in some cases a low contrast-to-noise ratio (CNR) prevents fast and reliable segmentation of the membranes. Synchrotron radiation µCT proved to be preferable for the visualisation of the small samples with very thin membranes, because of their high demands for spatial and contrast resolution. The best contrast was obtained by using synchrotron radiation µCT working in phase-contrast mode, leading to up to twice as high CNRs than the best conventional µCT results. The CNR of the synchrotron radiation µCT scans was sufficiently high enough to enable the construction of a 3D model by the means of semi-automatic segmentation of the membranous labyrinth. Membrane thickness was found to range between 2.7 and 36.3 µm. Hence, the minimal membrane thickness was found to be much smaller than described previously in the literature (between 10 and 50 µm).

Eardrum and columella displacement in single ossicle ears under quasi-static pressure variations.

Although most birds encounter large pressure variations during flight, motion of the middle ear components as a result of changing ambient pressure are not well known or described. In the present study, motion of the columella footplate and tympanic membrane (extrastapedius) in domestic chickens (Gallus gallus domesticus) under quasi-static pressure conditions are provided. Micro-CT scans were made of cadaveric heads of chickens under positive (0.25 kPa, 0.5 kPa, 1 kPa, and 1.5 kPa) and negative (-0.25 kPa, -0.5 kPa, -1 kPa, and -1.5 kPa) middle ear pressure. Both extrastapedius and columella footplate displacements show a non-linear S-shaped curve as a function of pressure indicating non-linear response characteristics of the middle ear components. The S-curve is also seen in mammals, but unlike in mammals, the lateral piston-like displacement of both the columella footplate and extrastapedius, which is caused by an increased middle ear pressure are smaller than the medial piston-like displacements, caused by a decreased middle ear pressure of the same magnitude. Columella footplate piston displacements are always smaller than the extrastapedius piston displacements, indicating the flexibility of the extracolumella. The cone-shape of the avian tympanic membrane with inverted apex in comparison to the mammalian tympanic membrane can cause the inverted shape of the pressure response curve.

Do high sound pressure levels of crowing in roosters necessitate passive mechanisms for protection against self-vocalization?

High sound pressure levels (>120dB) cause damage or death of the hair cells of the inner ear, hence causing hearing loss. Vocalization differences are present between hens and roosters. Crowing in roosters is reported to produce sound pressure levels of 100dB measured at a distance of 1m. In this study we measured the sound pressure levels that exist at the entrance of the outer ear canal. We hypothesize that roosters may benefit from a passive protective mechanism while hens do not require such a mechanism. Audio recordings at the level of the entrance of the outer ear canal of crowing roosters, made in this study, indeed show that a protective mechanism is needed as sound pressure levels can reach amplitudes of 142.3dB. Audio recordings made at varying distances from the crowing rooster show that at a distance of 0.5m sound pressure levels already drop to 102dB. Micro-CT scans of a rooster and chicken head show that in roosters the auditory canal closes when the beak is opened. In hens the diameter of the auditory canal only narrows but does not close completely. A morphological difference between the sexes in shape of a bursa-like slit which occurs in the outer ear canal causes the outer ear canal to close in roosters but not in hens.

Quasi-static and dynamic motions of the columellar footplate in ostrich (Struthio camelus) measured ex vivo.

The nature of the movement of the columellar footplate (CFP) in birds is still a matter of ongoing debate. Some sources claim that rocking motion is dominant, while others propose a largely piston-like motion. In this study, motions of the CFP are experimentally investigated in the ostrich using a post-mortem approach. For quasi-static loads, micro-CT scans of ostrich heads were made under positive and negative middle-ear pressures of 1 kPa. For dynamic loads, laser Doppler vibrometry was used to measure the velocity on multiple locations of the CFP as a function of excitation frequency from 0.125 to 4 kHz, and digital stroboscopic holography was used to assess the 1D full-field out-of-plane displacement of the CFP at different excitation frequencies. To expose the CFP in the experiments, measurements were made from the medial side of the CFP after opening and draining the inner ear. To determine the influence of the inner-ear load on CFP motions, a finite element model was created of the intact ostrich middle ear with inner-ear load included. For quasi-static loads, the CFP performed largely piston-like motions under positive ME pressure, while under negative ME pressure the difference between piston and rocking motion was smaller. For dynamic loads, the CFP motion was almost completely piston-like for frequencies below 1 kHz. For higher frequencies, the motions became more complicated with an increase of the rocking components, although they never exceeded the piston component. When including the inner-ear load to the model, the rocking components started to increase relative to the piston component when compared to the result of the model with unloaded CFP, but only at high frequencies above 1 kHz. In this frequency range, the motion could no longer be identified as purely piston-like or rocking. As a conclusion, the current results suggest that CFP motion is predominantly piston-like below 1 kHz, while at higher frequencies the motion becomes too complicated to be described as purely piston-like or rocking.

Deformation of avian middle ear structures under static pressure loads, and potential regulation mechanisms.

Static pressure changes can alter the configuration and mechanical behavior of the chain of ossicles, which may affect the acoustic transfer function. In mammals, the Eustachian tube plays an important role in restoring ambient middle ear pressure, hence restoring the acoustic transfer function and excluding barotrauma of the middle and inner ear. Ambient pressure fluctuations can be potentially extreme in birds and due to the simple structure of the avian middle ear (one ossicle, one muscle), regulation of the middle ear pressure via reflexive opening of the pharyngotympanic tube appears all the more important. In this study the deformations of the chicken (Gallus gallus domesticus) middle ear structures, as a result of middle ear pressure alterations, are quantified, using micro-CT scanning. It was experimentally tested whether reflexive opening of the pharyngotympanic tube to restore ambient middle ear pressure is present in chicken and mallard (Anas platyrhynchos) and whether this mechanism depends on sensing middle ear pressure indirectly via deformations of the middle ear components or sensing the middle ear pressure directly. A translation of the columella footplate was observed when middle ear pressure was kept at 1kPa and -1kPa relative to ambient pressure. Deformation of the tympanic membrane was larger than the columella footplate translation. Bending and deformation of the extracolumella was observed. Opening of the pharyngotympanic tube occurred at random pressure for both chicken and mallard when middle ear pressure was raised and lowered by 1.5kPa relative to ambient pressure. We also did not find a difference in middle ear venting rate when middle ear pressure was held constant at 0.5, 1, 1.5, -0.5, -1 and -1.5kPa for chickens and at 1, 2, 4, -1, -2 and -4kPa for mallards. As a result, no statement can be made about pressure within the avian middle ear being measured directly or indirectly. Our experiments do not support the presence of a short-loop reflexive control of pressure equilibration via the pharyngotympanic tube. However, it is still possible that triggering this loop requires additional sensorial input (e.g. visual, vestibular) or that it occurs voluntarily (being controlled at a higher brain level).

The effect of craniokinesis on the middle ear of domestic chickens (Gallus gallus domesticus).

The avian middle ear differs from that of mammalians and contains a tympanic membrane, one ossicle (bony columella and cartilaginous extra-columella), some ligaments and one muscle. The rim of the eardrum (closing the middle ear cavity) is connected to the neurocranium and, by means of a broad ligament, to the otic process of the quadrate. Due to the limited number of components in the avian middle ear, the possibilities of attenuating the conduction of sound seem to be limited to activity of the stapedius muscle. We investigate to what extent craniokinesis may impact the components of the middle ear because of the connection of the eardrum to the movable quadrate. The quadrate is a part of the beak suspension and plays an important role in craniokinesis. Micro-computed tomography was used to visualize morphology and the effect of craniokinesis on the middle ear in the domestic chicken (Gallus gallus domesticus). Both hens and roosters are considered because of their difference in vocalization capacity. It is hypothesized that effects, if present, of craniokinesis on the middle ear will be greater in roosters because of their louder vocalization. Maximal lower jaw depression was comparable for hens and roosters (respectively 34.1 ± 2.6° and 32.7 ± 2.5°). There is no overlap in ranges of maximal upper jaw elevation between the sexes (respectively 12.7 ± 2.5° and 18.5 ± 3.8°). Frontal rotation about the transversal quadrato-squamosal, and inward rotation about the squamosal-mandibular axes of the quadrate were both considered to be greater in roosters (respectively 15.4 ± 2.8° and 11.1 ± 2.5°). These quadrate rotations did not affect the columellar position or orientation. In hens, an influence of the quadrate movements on the shape of the eardrum could not be detected either; however, craniokinesis caused slight stretching of the eardrum towards the caudal rim of the otic process of the quadrate. In roosters, an inward displacement of the conical tip of the tympanic membrane of 0.378 ± 0.21 mm, as a result of craniokinesis, was observed. This is linked to a flattening and slackening of the eardrum. These changes most likely go along with a deformation of the extra-columella. Generally, in birds, larger beak opening is related to the intensity of vocalization. The coupling between larger maximal upper jaw lifting in roosters and the slackening of the eardrum suggest the presence of a passive sound attenuation mechanism during self-vocalization.

Sound attenuation in the ear of domestic chickens (Gallus gallus domesticus) as a result of beak opening.

Because the quadrate and the eardrum are connected, the hypothesis was tested that birds attenuate the transmission of sound through their ears by opening the bill, which potentially serves as an additional protective mechanism for self-generated vocalizations. In domestic chickens, it was examined if a difference exists between hens and roosters, given the difference in vocalization capacity between the sexes. To test the hypothesis, vibrations of the columellar footplate were measured ex vivo with laser Doppler vibrometry (LDV) for closed and maximally opened beak conditions, with sounds introduced at the ear canal. The average attenuation was 3.5 dB in roosters and only 0.5 dB in hens. To demonstrate the importance of a putative protective mechanism, audio recordings were performed of a crowing rooster. Sound pressures levels of 133.5 dB were recorded near the ears. The frequency content of the vocalizations was in accordance with the range of highest hearing sensitivity in chickens. The results indicate a small but significant difference in sound attenuation between hens and roosters. However, the amount of attenuation as measured in the experiments on both hens and roosters is small and will provide little effective protection in addition to other mechanisms such as stapedius muscle activity.