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The semicircular canals (SCCs) in the vestibular system can sense angular motion of the head, which performs a crucial role in maintaining the human's sense of balance. The different spatial orientations of the head affect the response of human SCCs to rotational movement. In this study, we combined the numerical model of bilateral human SCCs with vestibulo-ocular reflex experiments, and quantitatively investigated the responses of SCCs to constant angular acceleration when the head was in different left-leaning positions, including the head tilted 0°, 15°, 30°, 45°, 60°, 70°, 80°, and 90° to the left. The results showed that the vertical nystagmus slow-phase velocity (SPV) and the corresponding maximal cupula shear strain at the crista surface rose with an increase in the left-leaning angle of the head, reached a maximum at the position of the head tilted approximately 70° to the left, and then decreased gradually. Both the horizontal nystagmus SPV and the corresponding maximal cupula shear strain at the crista surface were the largest under the position of the head tilted 0° to the left, and decreased gradually as the left-leaning angle of the head increased. The numerical results of cupula shear strain at the crista surface in bilateral SCCs can quantitatively explain the combined effects of each SCC's excitation or inhibition on volunteers' nystagmus SPV under different head positions. In addition, a fluid-structure interaction investigation revealed that different left-leaning head positions changed the endolymphatic pressure gradient distribution in SCCs, which determined the transcupular pressure, cupula shear strain at the crista surface, and nystagmus SPV.

Mechanosensory hair cells in the chicken inner ear are innervated by bipolar afferent neurons of the statoacoustic ganglion (SAG). During development, individual SAG neurons project their peripheral process to only one of eight distinct sensory organs. These neuronal subtypes may respond differently to guidance cues as they explore the periphery in search of their target. Previous gene expression data suggested that Slit repellants might channel SAG neurites into the sensory primordia, based on the presence of robo transcripts in the neurons and the confinement of slit transcripts to the flanks of the prosensory domains. This led to the prediction that excess Slit proteins would impede the outgrowth of SAG neurites. As predicted, axonal projections to the primordium of the anterior crista were reduced 2–3 days after electroporation of either slit1 or slit2 expression plasmids into the anterior pole of the otocyst on embryonic day 3 (E3). The posterior crista afferents, which normally grow through and adjacent to slit expression domains as they are navigating towards the posterior pole of the otocyst, did not show Slit responsiveness when similarly challenged by ectopic delivery of slit to their targets. The sensitivity to ectopic Slits shown by the anterior crista afferents was more the exception than the rule: responsiveness to Slits was not observed when the entire E4 SAG was challenged with Slits for 40 h in vitro. The corona of neurites emanating from SAG explants was unaffected by the presence of purified human Slit1 and Slit2 in the culture medium. Reduced axon outgrowth from E8 olfactory bulbs cultured under similar conditions for 24 h confirmed bioactivity of purified human Slits on chicken neurons. In summary, differential sensitivity to Slit repellents may influence the directional outgrowth of otic axons toward either the anterior or posterior otocyst.

Noise over-stimulation will induce or influence molecular pathways in the cochlea; one approach to the identification of the components of these pathways in the cochlea is to examine genes and proteins that change following different types and levels of stress. Quantitative reverse transcription polymerase chain reaction provides a method to look at differential expression of genes in the acoustic stress response. By using this technique we have revealed a down-regulation of the level of otospiralin mRNA in the cochlea of guinea pigs after white noise over-stimulation for 2 h at 108 dB SPL. Otospiralin represents an inner ear specific protein found in fibrocytes of spiral limbus and spiral ligament in the cochlea, and some regions of the vestibule as the stroma underlying the utricle and crista sensory epithelia and the subepithelial layer of the walls of semicircular canals and maculae. It has been recently reported that transient down-regulation of otospiralin in guinea pigs causes vestibular syndrome and deafness. Our results suggest a possible role of this gene in response to acoustical stress, although the exact mechanism remains to be resolved.

The present study describes the localization and distribution of putative ecto-nucleoside-triphosphate-diphosphohydrolases in the frog semicircular canals. These enzymes provide the terminating mechanism of adenosine-5'-triphosphate (ATP) signalling. The localization of the ATP hydrolysis was mapped ultracytochemically using a one-step cerium citrate reaction. Electron-dense precipitates, indicating ecto-adenosine-triphosphatase (ecto-ATPase) activity, were found at the outer surface of plasma membranes of crista hair cells and supporting cells of the sensory epithelium, transitional cells and undifferentiated cells of the ampullar wall and dark cells constituting the secretory epithelium. Non-sensory cells of the ampulla usually exhibited reaction deposits at the level of both apical and basolateral membranes coming into contact with the endolymph and the perilymph respectively, while cells constituting the sensory epithelium showed evident differences in relation to their position. Hair cells and supporting cells of the peripheral regions exhibited clear reaction products both at the level of apical and basolateral membranes, while those of the isthmus region showed abundant reactivity only at the level of their apical membranes. Of particular interest was the observation that hair cell stereocilia exhibited an abundant ecto-ATPase activity, thus suggesting a possible colocalization of enzymatic sites with purinergic receptors and mechanotransduction channels. This strategic expression of ecto-ATPase sites could provide a rapid mechanism of ATP removal able to rapidly restore the sensitivity of transduction channels. In conclusion, the widespread distribution of ecto-ATPase sites at the level of sensory and non-sensory cells of the frog semicircular canals suggests that ATP may have a key role in controlling vestibular function.

The list of distinguishing morphological features of hair cells includes: Type I and Type II afferent innervation, and length, shapes and arrangements of stereo- and kinocilia. We now add to this list the shapes of the hair cells themselves and their placement within the mechanosensory organ, in this case the semicircular canal. Although hair cells of the crista ampullaris of the frog are only of Type II they may now be further classified into three sub-groups according to shape: club-, cigar- and pear-shaped. The cigar- and club-shaped hair cells are each about 40% while the pear-shaped cells are about 20% of the total numbers of hair cells in the crista. The differently-shaped hair cells also distribute differently along the crista. The cigar- and club-shaped are more-or-less uniformly distributed with somewhat higher concentrations at the ends of the crista than in the center. The pear-shaped hair cells, on the other hand, are mostly concentrated toward the center of the crista. This distribution of the pear-shaped hair cells, and their shape is reminiscent of the distribution of calyceal endings (Type I hair cell) in the cristae of amniotes [Goldberg et al., Hear. Res. 49, 89–102 (1990) in Chinchilla; Fernandez et al., Soc. Neurosci. Abstr. 17, 312 (1991) in Monkey]. There are some quantitative differences between hair cells of the same shape but from different portions of the crista. For instance, pear-shaped hair cells of the center are generally of greater cross-sectional area than those of the ends. Also, club-shaped hair cells are taller in the slopes of the crista than they are in the center. Lastly, hair cells of all three shapes have smaller cross-sectional areas after isolation from the crista as, for example, in preparation for patch-clamping. The greatest of these areal differences is seen in the pear-shaped hair cells. Thus, the emerging picture of the hair cell of the semicircular canal when considering both morphology and physiology is one of diversity and complexity suggesting a role in signal processing of greater importance than previously appreciated.

Vascular change has been proposed as an etiological factor in inner ear aging and in several inner ear disorders. Moreover, some successful medical management of the episodic vertigo and tinnitus associated with Ménière's disease has been directed toward pharmacologically increasing blood flow, changing vascular permeability or ion homeostasis. While there are many studies of cochlear capillary morphology and blood flow, there are very few examining these variables in the vestibular system and none with respect to aging. The purpose of this study was to examine the rat posterior canal ampullary crista for age-related changes in blood flow and capillary morphology. By combining stereological techniques with microsphere injection, we have determined that in the rat posterior canal crista there is a statistically significant age-related decrease in blood flow (75%), mean capillary diameter (31%), and volume fraction of capillary lumen (31%). There is also an overall 18% decrease in the volume of the ampullary crista, a 72% decrease in blood flow/unit volume and a 36% increase in capillary length/unit volume. There were no significant changes in the capillary surface area/unit volume, the absolute capillary length, or the absolute capillary surface area. These data suggest impaired blood flow and degenerative loss of the ampullary crista may be relate to impaired end organ function.

There are three kinds of afferent terminations in the cristae ampullares. Calyx units innervate a few neighboring type I hair cells. Bouton units contact several type II hair cells. Dimorphic units innervate both kinds of receptors. Axon diameters are largest for calyx fibers and smallest for bouton fibers. Dimorphic units supply all parts of the sensory epithelium. Calyx units are confined to the central zone of the crista and bouton units to its peripheral zone. Intra-axonal labeling was used to determine the innervation patterns of physiologically characterized afferents. Calyx units are irregularly discharging. Dimorphic units in the central zone have a more irregular discharge than those in the peripheral zone. Bouton units, which have also been identified by their slow conduction velocities, are regularly discharging. An afferent's discharge regularity, sensitivity to externally applied galvanic currents and response dynamics are more closely related to its epithelial location than to its branching pattern or to the types and number of hair cells it contacts. Of the various discharge properties studied, only the rotational gains seemed closely related to terminal morphology. Afferents innervating the central and peripheral zones differ in their innervation patterns and discharge properties. A preliminary ultrastructural study indicates that there also are regional variations in synaptic organization. Type II hair cells in the peripheral zone are contacted by many more afferent boutons than those in the central zone. Individual central boutons have multiple ribbon synapses with type II hair cells, whereas each peripheral bouton usually has a single synaptic contact. Synapses between type II hair cells and calyx endings are common centrally, but not peripherally. Two synaptic features did not vary regionally: 1) type I hair cells usually make 10-20 ribbon synapses with their calyx endings; and 2) each type II hair cell is contacted by 2-6 efferent endings. The number of efferent boutons in contact with each calyx ending declines slightly from the peripheral zone to the central zone. Reciprocal synapses were rare.

The auditory organs of the tettigoniid are located just below the femoral tibial joint in the forelegs. Structurally each auditory organ consists of a tonotopically organized crista acustica and intermediate organ and associated sound conducting structures; an acoustic trachea and two lateral tympanic membranes located at the level of the receptor complex. The receptor cells and associated satellite structures are located in a channel filled with hemolymph fluid. The vibratory response characteristics of the tympanic membranes generated by sound stimulation over the frequency range 2-40 kHz have been studied using laser vibrometry. The acoustic trachea was found to be the principal structure through which sound energy reached the tympana. The velocity of propagation down the trachea was observed to be independent of the frequency and appreciably lower than the velocity of sound in free space. Structurally the tympana are found to be partially in contact with the air in the trachea and with the hemolymph in the channel containing the receptor cells. The two tympana were found to oscillate in phase, with a broad band frequency response, have linear coherent response characteristics and small time constant. Higher modes of vibration were not observed. Measurements of the pattern of vibration of the tympana showed that these structures vibrate as hinged flaps rather than vibrating stretched membranes. These findings, together with the morphology of the organ and physiological data from the receptor cells, suggest the possibility of an impedance matching function for the tympana in the transmission of acoustic energy to the receptor cells in the tettigoniid ear.

The structure of the complex tibial organs in the fore-, mid-, and hindlegs of the bushcricket Polysarcus denticauda (Tettigoniidae, Phaneropterinae) is described comparatively. As is common for bushcrickets, in each leg the tibial organs consist of the subgenual and intermediate organs and the crista acustica. Only in the forelegs are sound-transmitting structures present. They consist of the spiracle, acoustic trachea, and two tympana; the latter are not protected by tympanal covers. The tympana in P. denticauda are extremely thick, not only bordering the two tracheal branches to the outside but also forming the outer wall of the hemolymph channel. The morphology of the tracheae in the mid- and hindlegs is significantly different, causing structural differences, especially in dimensions of the hemolymph channel. The number of scolopidia of the crista acustica of the foreleg is extremely high for a bushcricket. Approximately 50 receptor cells were found, about half of them being located in the distal quarter of the long axis of this organ. Some of the receptors are positioned in parallel on the dorsal wall of the anterior tracheal branch. The number, morphology and dimensions of the scolopidia within the crista acustica of the mid- and hindlegs differ significantly from those of the forelegs, decreasing in both legs to eight and seven receptor cells, respectively. Although the dimensions of the subgenual and intermediate organs are considerably larger in the mid- and hindlegs, the number of receptor cells is approximately the same in the different legs, being somewhat higher in both receptor organs than in those of many other bushcricket species studied previously.

Two experiments were conducted to study the ototoxic effects of local gentamicin (GM) administration and the subsequent hair cell (HC) regeneration process in the chinchilla cristae ampullares (CA). In the first experiment, 3 different doses of GM (0.1, 0.2 and 1.2 mg) were administered by surgical implantation of GM-soaked Gelfoam® pledgets in the perilymphatic space in the otic capsule of the left superior semicircular canal. The CA was histologically processed for light-microscopic examination. In the second experiment, 6 groups of 2 chinchillas each were treated with 0.1 mg of GM. To document cell proliferation and HC regeneration, Alzet® micro-osmotic pumps were implanted in each chinchilla to deliver bromodeoxyuridine (BrdU) at 125 μg/h for 1 week. Chinchillas were subsequently killed at 1 and 4 days and 1, 2, 4 and 8 weeks post-treatment (PT). The CA was processed for light microscopy and BrdU immunocytochemistry. In the first experiment the smallest dose produced damage restricted to HCs alone, while the medium and large doses produced severe damage in the sensory epithelium, including supporting cells and HCs. Results in the second experiment demonstrated that at 1 and 4 days PT and HCs showed extensive damage, including clumping of nuclear material. By 4 days PT the supporting cell nuclei lost their monolayer configuration. Calyceal terminals appeared empty, and vacuolized remnants of nerve calyces were evident in the basal portion. At 1 week PT complete disappearance of HCs from the sensory epithelium was evident, and there was cytoplasmic extrusion into the endolymphatic space. At 2 weeks PT there was complete HC loss, the supporting cell nuclei were scattered randomly in the crista, and the nerve fibers were retracted from the sensory epithelium. At 4 weeks PT there was evidence of sensory epithelium repair and HC regeneration. Short cells resembling type-II HCs were evident in the surface of the sensory epithelium. At 8 weeks PT the number of HCs increased in a uniform fashion on the surface of the sensory epithelium, and the supporting cell nuclei were realigned on the basal membrane. Nerve fibers with growth cones penetrated the basal membrane. Supporting cell proliferation was evident by the presence of mitotic figures and BrdU immunoreactivity in the chromatin material of dividing cells at 2 weeks PT. The labeling was more evident in newly formed cells at 4 and 8 weeks PT. These results demonstrate that in chinchillas the vestibular organs have the capacity of self-repair and the process includes HC regeneration after local administration of GM. The overall process involves changes in different cells in the sensory epithelium and neural elements, all of which show modifications with an orderly pattern.

The distribution of GABA A receptor-like immunoreactivity in the hamster, rat, and mouse crista ampullaris was determined by use of a monoclonal antibody to the β2 and β3 subunits of the GABA A receptor. In the crista ampullaris, punctate staining was seen associated with the calyces surrounding vestibular type I hair cells. Afferent nerve fibers approaching the hair cell layer were often observed to be immunoreactive. Hair cells, supporting cells, and cells in the transitional and dark cell regions were not immunoreactive. The distribution of staining of calyces appeared to be relatively uniform in all regions (crest and slope) of the crista. In addition, cell bodies located in the vestibular ganglion were immunoreactive. The association of GABA A receptor-like immunoreactivity with the afferent nerve calyx and cell body of the vestibular ganglion cells suggests that GABA may act to modify afferent nerve transmission at the calyceal afferent nerve ending.

The bushcricket species Decticus albifrons , Decticus verrucivorus and Pholidoptera griseoaptera (Tcttigoniidae) belong to the same subfamily ( Decticinae ) but differ significantly in body size. In spite of the great differences in the dimensions of the forelegs, where the auditory organs are located, the most sensitive range of the hearing threshold lies between 6 and 25 kHz in each case. Only in the frequency range from 2 to 5 kHz and above 25 kHz, significant differences are present. The anatomy of the auditory receptor organs was compared quantitatively, using the techniques of semi-thin sectioning and computer-guided morphometry. The overall number of scolopidia and the length of the crista acustica differs in the three species, but the relative distribution of scolopidia along the crista acustica is very similar. Additionally, the scolopidia and their attachment structures (tectorial membrane, dorsal tracheal wall, cap cells) are of equal size at equivalent relative positions along the crista acustica. The results indicate that the constant relations and dimensions of corresponding structures within the cristae acusticae of the three species are responsible for the similarities in the tuning of the auditory thresholds.

Hair cells isolated from the crista ampullaris of the frog (Rana pipiens) remained viable for up to 5 h and were studied using whole cell voltage- and current clamp recordings. Morphological characteristics of isolated crista hair cells were compared with hair cells studied in situ using light- and electron microscopy. While other labyrinthine hair cells such as mammalian inner and outer hair cells of the cochlea, saccular hair cells of the frog, and cochlear hair cells of the turtle typically have a cylindrical shape, the crista hair cells in the frog are predominantly bulbous, having a thin elongated trunk projecting from a spherical base just large enough to enclose the nucleus. This shape correlates well with the compressed packing configuration of hair cells of the crista ampullaris observed in situ in the histological material. The support cells often failed to separate adjacent hair cells, particularly the apical ends of the hair cells. Maximal cell density on the sensory epithelial ridge appears to be achieved by this arrangement. The mean resting membrane potential (Vz) of isolated crista hair cells was −44.8 mV. Cells with smooth surfaces and apparent opacity had the most negative Vz potentials. As the cells appeared to deteriorate, there was development of transparency and cell surface granulation. Such cells had more positive initial Vz values. Cells with Vz values more positive than −15 mV exhibited a distinct, contoured nucleus. Cells lacking these indicators of deterioration were characterized by input resistances of 1.9 ± 0.31 GΩ and membrane time constants of 13 ± 2.5 ms. A large complex outwardly rectifying current was identified which was abolished by substituting Cs+ for K+ in the internal solution. The outward K+ current had two major components: a fast tetraethylammonium (TEA)-insensitive, voltage dependent I(A)-type current which showed voltage dependent inactivation; and a TEA sensitive current which had characteristics of a calcium dependent IK(Ca)-type current. Transient changes (20 ms duration) in membrane potential mimicking that which could be produced by the transduction current during cilial displacement potently modulated the I(A) current. Depolarizing current pulses of greater than 63800 pA were required to elicit membrane voltage oscillations. The resulting membrane potential offset of at least 40 mV is well beyond the magnitude of hair cell receptor potentials making it unlikely that these oscillations would play a role in enhancing frequency selectivity. The presence of a long membrane time constant, a membrane stabilizing I(A) conductance and a relatively small IK(Ca) conductance explains the difficulty in eliciting oscillations in crista hair cells and may be related to the optimization of sensitivity of the hair cells for detecting cupular movement during angular acceleration.

The vestibular organs of young and very old C57BL/6NNia (B6) mice were compared by light and electron microscopy. Hair cell density decreased an average of 14% in the utricle, 19% in the saccule and posterior crista, 23% in the horizontal crista, and 24% in the anterior crista. Hair cell size remained the same throughout the mouse's life span as did the ratio of Type I to Type II hair cells. The most apparent sign of advanced age was dense inclusions found in sensory and supporting cells. Although small inclusions were present at five weeks, by 29 months, additional, larger forms appeared. An unusual melanin-like form was characteristic of old Type I hair cells. Synaptic morphology and synaptic bodies were well preserved even in very old B6 mice. Elongated bars were common in Type I hair cells and spheroid synaptic bodies were the most common form in Type II hair cells. Large clusters of synaptic bodies occurring in both young and old mice were seen only in Type I hair cells. Although the B6 strain suffers from genetically determined early cochlear degeneration, it does not experience early degeneration of the peripheral vestibular organs.

The aim of the investigation was to obtain quantitative measures of mechanical filter properties of hair cell sensory hairs which were not restricted to move by auxiliary structures. The specimen used was the crista ampullaris of the frog, dissected free and positioned in a fluid-filled chamber where it could be viewed with differential interference contrast optics. The sensory hairs were displaced by a brief jet of frog Ringer's solution from a specially constructed microsyringe apparatus. The velocity of the jet could be stepwise controlled and was determined by measuring the speed of motion of 3 micron plastic beads propelled with the jet across the microscopic field. The stereocilia displacements were recorded on 16 min film, and both the angle of deflection and the time for the return to the resting position were measured on the film. It was found that after displacement the sensory hairs returned to the upright position by elastic properties in the hinge region at their insertion point in the cuticular plate. Sensory hairs differed in their speed of return so that some had fast time constants, others quite slow ones. This was correlated to a difference in structural development of the stereocilia, fast sensory hairs having thick and tall stereocilia, slow ones having thin and short stereocilia. The various bundle types were identified in the scanning electron microscope and their distribution on the crista was mapped. This was found to match the distribution of nerve fibres with different functional properties. It is concluded that sensory hair cells can differ in their mechanical filter properties as a result of the structural arrangement of their stereocilia and in accordance with functional demands.