Comparative study of visuo-vestibular conditioning in Lymnaea stagnalis.

In this review, we compare the current understanding of visuo-vestibular conditioning in Hermissenda crassicornis and Lymnaea stagnalis on the basis of behavioral, electrophysiologic, and morphologic studies. Paired presentation of a photic conditioned stimulus (CS) and an orbital rotation unconditioned stimulus (US) results in conditioned escape behavior in both species. In Hermissenda, changes in excitability of type B photoreceptors and morphologic modifications at the axon terminals follow conditioning. Caudal hair cells, which detect mechanical turbulence, have reciprocal inhibition with type B photoreceptors. In Lymnaea, the interaction between photoreceptors and hair cells is dependent on statocyst location. Furthermore, the organization of the Lymnaea eye is complex, with more than 100 photoreceptors distributed in a uniquely folded retina. Although the optimal conditions to produce long-term memory (memory persistent for >1 week) are almost identical in Hermissenda and Lymnaea, physiologic and morphologic differences suggest that the neuronal mechanisms underlying learning and memory are distinct.

Mechanical properties and consequences of stereocilia and extracellular links in vestibular hair bundles.

Although knowledge of the fine structure of vestibular hair bundles is increasing, the mechanical properties and functional significance of those structures remain unclear. In 2004, Bashtanov and colleagues reported the contribution of different extracellular links to bundle stiffness. We simulated Bashtanov's experimental protocol using a three-dimensional finite element bundle model with geometry measured from a typical striolar hair cell. Unlike any previous models, we separately consider two types of horizontal links: shaft links and upper lateral links. Our most important results are as follows. First, we identified the material properties required to match Bashtanov's experiment: stereocilia Young's modulus of 0.74 GPa, tip link assembly (gating spring) stiffness of 5,300 pN/microm, and the combined stiffness of shaft links binding two adjacent stereocilia of 750 approximately 2,250 pN/microm. Second, we conclude that upper lateral links are likely to have nonlinear mechanical properties: they have minimal stiffness during small bundle deformations but stiffen as the bundle deflects further. Third, we estimated the stiffness of the gating spring based on our realistic three-dimensional bundle model rather than a conventional model relying on the parallel arrangement assumption. Our predicted stiffness of the gating spring was greater than the previous estimation.

Quantitative analysis of stereociliary arrays on vestibular hair cells.

We have developed a method for quantifying the number, spacing, and distribution of stereocilia on the apical surface of hair cells using spatial autocorrelation analysis and statistics for directional data. Here, we illustrate the method using idealized hair bundles, and we apply it to scanning micrographs of turtle hair cells from the utricle and posterior canal, and to freeze-fracture preparations of bullfrog saccule. The analysis suggests three common features of stereociliary bundles. First, bundle geometries form a continuum from 'loose' to 'tight' rather than two distinct groups. Second, interciliary spacing along the three hexagon axes is not equal; spacing is usually widest along the hexagon axis closest to the bundle's axis of bilateral symmetry (the presumptive excitatory axis). Third, spacing between stereocilia changes with distance from the kinocilium. All three features will influence predictions of the tip link tensions that accompany bundle deflection.

Evolutionary trends in the organization of the vertebrate crista ampullaris.

Intraaxonal labeling studies in the toadfish, frog, turtle, and chinchilla suggest broad evolutionary trends in the vertebrate crista ampullaris. The crista of anamniotes (fish, amphibians) contains type II hair cells innervated by bouton afferents and is longitudinally organized. Type I hair cells are first seen in reptiles and birds, where they are confined to a central zone and are innervated by calyx and dimorphic afferents. The central zone is surrounded by a peripheral zone containing only type II hair cells innervated by bouton afferents. Results in the turtle suggest that the peripheral zone in reptiles and birds is organized similarly to the entire anamniote crista. The turtle central zone finds no parallel in anamniotes but resembles the mammalian central zone in its structure and afferent physiology. With the advent of a central zone in reptiles, a concentric organization is superimposed on a linearly organized peripheral zone. The mammalian crista, in contrast, has an entirely concentric organization. This may be related to the extension of the neuroepithelium further down the slopes of the crista in mammals than in other vertebrates and to the distribution of type I hair cells throughout the mammalian neuroepithelium.

Hair cells in mammalian utricles.

Two morphological classes of mechanosensory cells have been described in the vestibular organs of mammals, birds, and reptiles: type I and type II hair cells. Type II hair cells resemble hair cells in other organs in that they receive bouton terminals from primary afferent neurons. In contrast, type I hair cells are enveloped by large cuplike afferent terminals called calyces. Type I and II cells differ in other morphological respects: cell shape, hair bundle properties, and more subtle ultrastructural features. Understanding the functional significance of these strikingly different morphological features has proved to be a challenge. Experiments that correlated the response properties of primary vestibular afferents with the morphologies of their afferent terminals suggested that the synapse between the type I hair cell and calyx ending is lower gain than that between a type II hair cell and a bouton ending. Recently, patch-clamp experiments on isolated hair cells have revealed that type I hair cells from diverse species have a large potassium conductance that is activated at the resting potential. As a consequence, the voltage responses generated by the type I hair cells in response to injected currents are smaller than those generated by type II hair cells. This may contribute to the lower gain of type I inputs to primary afferent neurons. Studies of neonatal mouse utricles show that the type I-specific potassium conductance is not present at birth but emerges during the first postnatal week, a period of morphological differentiation of type I and type II hair cells.

Age-related changes of the globular substance in the otoconial membrane of mice.

The globular substance, which occurs in the vestibular macula as a precursor of otoconia, was examined in aged mice in comparison with young adult mice. Dissected otoconial membrane from the utricular macula of C57BL/6J mice was loaded with fluo-3-AM, and directly observed under a confocal laser scanning microscope. Internal free Ca2+ concentration ([Ca2+]i) of the globular substance was determined through in situ calibration performed by superfusion with ionomycin and Mn2+. Total area of the otoconial membrane, average diameter of the globular substance, and [Ca2+]i showed no significant differences between young adult and aged groups. However, the number of globular substances in young adult mice was significantly larger than those of aged mice. These results suggest a reduced rate of otoconial formation in the aged vestibule, which would result in the sparseness of otoconia in the aged vestibule and lead to balance disorders commonly seen in elderly persons.

Convergent evolution of the vestibular organ in the subterranean mole-rats, Cryptomys and Spalax, as compared with the aboveground rat, Rattus.

The membranous labyrinth of the vestibular organ (examined in toto) in two unrelated species of subterranean rodents, Cryptomys sp. from Zambia and Spalax ehrenbergi from Israel, was in many parameters (streamline length, curvature radius, and cross-sectional area of the lumen) relatively or even absolutely (especially the cross-sectional area) larger than in the laboratory Norway rat. The mechanical sensitivity of the vestibular organ (estimated according to the mathematical model of Oman et al., [1987] Acta. Otolaryngol (Stockh.) 103:1-13) was similar in both subterranean rodent species and significantly higher than that in the laboratory rat. The most pronounced differences in morphometry and the resulting mechanical sensitivity between the subterranean forms and the rat occurred in the lateral (i.e., phylogenetically and ontogenetically most recent and presumably most plastic) semicircular duct. The area of the sensory epithelia, and density and total numbers of vestibular receptors, were estimated on surface specimens for both maculae and for all three cristae for all three species. While the density of hair cells in comparable sensory epithelia was similar in all three species, the sensory area and thus, also, the total receptor counts were significantly larger in both subterranean forms. The peripheral vestibular organ in subterranean rodents is, in comparison to a generalized aboveground dwelling form, i.e., the rat, progressively specialized, and in any case cannot be denoted as degenerate.

Hyaluronic acid as a molecular filter and friction-reducing lubricant in the human inner ear.

Immunofluorescence for hyaluronic acid occurred intracellularly in morphologically highly specialized areas in the adult human inner ear, for instance in the cuticular plates of all types of hair cells, at the apposition between outer hair cells and Deiter's cell bodies and in the near-surface area of Hensen's cells. The cytoskeletal organization in these regions is characterized by tightly packed filamentous proteins. Under physiological stimulus these regions undergo micromechanical change, either actively moving (force generation) or passively vibrating with changes in elasticity. Hyaluronic acid might therefore act as a friction-reducing molecular lubricant. In the lateral wall of the cochlea an accumulation of hyaluronic acid occurred in the loose connective tissue of the spiral ligament, in particular close to the stria vascularis. Due to its complex molecular network, hyaluronic acid offers considerable resistance to bulk flow of water and may exclude molecules. The basal cell region of the stria vascularis is thus given additional support to minimize (seal?) the stria vascularis towards all other areas except the endolymphatic space. Here, hyaluronic acid could act as a molecular filter.

Calbindin-D28K localization in the primate inner ear.

The distribution of one of the calcium-binding proteins, calbindin-D28K (CB-D28K), was studied in the adult human and squirrel monkey inner ear by means of immunocytochemical methods. Inner and outer hair cells in the organ of Corti and vestibular hair cells showed CB-D28K immunoreactivity, though some vestibular hair cells were devoid of immunoreactivity. In the spiral and vestibular ganglion, immunoreactive cells were found in both the squirrel monkey and human. The present results indicate that CB-D28K is localized within afferent neuronal components in these sensory organs and may regulate Ca++ levels for optimal neurotransmission in the primate auditory and vestibular systems. This study also provides evidence of two nonneuronal localizations of CB-D28K in the squirrel monkey. Subpopulations of fibrocytes in the spiral ligament and vestibular end organs were enriched with CB-D28K, suggesting that these cells are possibly equipped with the function to regulate Ca++ concentration in the perilymphatic fluid. In the maculae, many CB-D28K-immunoreactive particles were found in the otoconial membrane, indicating that CB-D28K may participate in the formation of otoconia.

Microtubule-associated proteins in adult human sensory organs.

The distribution of microtubule-associated proteins MAP-1 and MAP-2 was analysed with immunomorphological techniques in the serially sectioned adult human membranous labyrinth. In the organ of Corti, monoclonal antibodies to MAP-1 did not stain. Positivity for MAP-2 occurred in the entire outer hair cell cytoplasm, in the inner hair cells (?), in the nerve fibres and in the cytoplasm of epithelial cells of the spiral prominence. In addition, staining for MAP-2 was identified in many (but not all) cells or Reissner's membrane. Immunofluorescence for MAP-1 occurred in the supporting cells of the cristae and maculae interpreted to be localized in the apical region adjacent to the sensory cells. Thus, the distribution of MAP-1 and MAP-2 in the adult human membranous labyrinth was the same as described for several animal species with regard to the cochlea. In contrast to such a pattern, both MAP-1 and MAP-2 were identified in the human vestibular organs, thus identifying a subpopulation of centrally located nerve calyces and possibly also the apical portion of vestibular hair cells.

Morphological features of different regions in frog crista ampullaris (Rana esculenta).

The cellular organization of different regions of the crista epithelium from the frog posterior semicircular canal was studied by light, transmission and scanning microscopy. The sensory epithelium consists of hair cells surrounded by supporting cells and basal cells located close to the basement membrane. Three types of hair cells, namely club-like, cylindrical and pear-like cells differentially distributed along the crista could be recognized on the basis of their shape. Club-like cells are located only in the peripheral regions, cylindrical cells both in the central and in the peripheral regions, and pear-like cells appear segregated into the intermediate regions. Sensory cells of the central region are characterized by a ciliary apparatus consisting of stereocilia usually shorter--and in some cases less numerous--than those of cells of the other regions. The presence of large evaginations of the apical membrane of hair cells and of several vesicles of microexocytosis demonstrates that receptor cells have a considerable secretory activity. This secretory activity is also proven by the presence in the supranuclear region of hair cells of numerous Golgi complexes. Moreover, the presence of two kinds of Golgi complexes, one constituted by dilated cisternae containing a moderately electron-dense material and the other made up of flattened electron-transparent cisternae, suggests a diversified secretion of material by the hair cells. This heterogeneous material may provide substances important for cupula formation and the composition of the endolymph.

In vivo regeneration of vestibular hair cells of guinea pig.

Recent studies have shown that inner ear sensory cells may regenerate in mammals as well as in birds. In this in vivo study we investigated the regeneration of vestibular hair cells in guinea pigs after gentamycin intoxication, and found that the vestibular sensory epithelia reproduced new hair cells 30 days after degeneration, due to 30 consecutive days of gentamycin treatment. From the regeneration pattern of sensory epithelia, the supporting cells were identified as the progenitors of hair cells.

Morphometric studies of type I and type II hair cells in the gerbil's posterior semicircular canal crista.

The existence of separate subtypes of type I vestibular hair cells according to morphological criteria in situ was investigated. Gerbils were anesthetized and perfused with mixed aldehydes. The crista ampullaris of the posterior canal was dissected, fixed in osmium, dehydrated, and embedded in epon. Five-micron sections were cut orthogonal to the long axis of each crista. Measurements were made on camera lucida drawings of individual cells located in the apical, middle, and basal 1/3 of the crista. Measurements for each hair cell included the circumference, greatest width of the body, length, width of the apical surface (cuticular plate region, P), width at narrowest portion of the neck (NW), neck width to plate ratio (NPR), length at a point 2 times NW from the apical surface (L2N). Type I hair cells were subgrouped into three classes (long -1, intermediate -i, and short -s) based on a subjective determination of neck length. Statistical comparisons were made between type I (n = 612) and type II (n = 74) hair cells and the type I subtypes (l, i, s). Statistically significant differences were found between type I and II hair cells for NPR, width, and length, but not perimeter. Thus, as in pigeons, NPR distinguishes type I and type II hair cells in the gerbil crista. While type I hair cells are wider and longer than type IIs, the circumference is the same, due to the restricted neck in type I hair cells. The L2N statistic separates three subtypes of type I hair cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytoskeletal organization of the vestibular sensory epithelia: saponin perfusion method for observing intracellular structures by scanning electron microscopy.

The cytoskeletal organization of the guinea pig vestibular sensory epithelial cells were investigated by the use of saponin perfusion method using scanning electron microscopy. The skeletal framework of a cell is composed of thin (actin or intermediate filaments) and thick filaments (microtubules). The membrane bound organelles such as nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, etc. were also well demonstrated. This made it possible to investigate the three-dimensional structures of cytoskeletons as well as their complex interactions with various membranes bound organelles. It is therefore suspected that this technique may provide us further information about distribution, topographic relationships, and the functional role of cytoskeletons.

Effects of microgravity on vestibular ontogeny: direct physiological and anatomical measurements following space flight (STS-29).

Does space flight change gravity receptor development? The present study measured vestibular form and function in birds flown as embryos for 5 days in earth orbit (STS-29). No major changes in vestibular gross morphology were found. Vestibular response mean amplitudes and latencies were unaffected by space flight. However, the results of measuring vestibular thresholds were mixed and abnormal responses in 3 of the 8 flight animals raise important questions.

Toward modeling a dynamic biological neural network.

Mammalian macular endorgans are linear bioaccelerometers located in the vestibular membranous labyrinth of the inner ear. In this paper, the organization of the endorgan is interpreted on physical and engineering principles. This is a necessary prerequisite to mathematical and symbolic modeling of information processing by the macular neural network. Mathematical notations that describe the functioning system were used to produce a novel, symbolic model. The model is six-tiered and is constructed to mimic the neural system. Initial simulations show that the network functions best when some of the detecting elements (type I hair cells) are excitatory and others (type II hair cells) are weakly inhibitory. The simulations also illustrate the importance of disinhibition of receptors located in the third tier in shaping nerve discharge patterns at the sixth tier in the model system.