A transmission and scanning electron microscopic study of the saccule in five species of catfishes

The sacculi of five species of catfishes were studied by transmission and scanning electron microscopy. In four species, the sagitta exhibited a multifluted anterior part and a tapered posterior part; in Corydoras aeneus, however, the fluted part was absent, and a vertical component extended dorsally to terminate near the opening of the transverse canal. In all species, the otoliths had a laminar structure. An otolithic membrane was present, and hair cell bundles projected into cavities on the macular surface of the membrane. Attachments of the otolithic membrane to the neuroepithelium included short extensions of the membrane to the tallest components of the hair cell bundles of the peripheral cells and more delicate connections to the kinocilium and taller stereocilia of central cells; in addition, attachments to the microvilli of supporting cells were present. In both hair cells and supporting cells single microtubules and bundles of microtubules were present; the bundles had an orderly arrangement and were associated with cytoplasmic densities surrounding the desmosomes. The hair cells were innervated by both afferent and efferent nerve endings. Studies of the polarization of the hair cells in all species (except C. aeneus) showed that there was a single longitudinal axis that divided dorsally polarized cells from those oriented ventrally. In Doras spinosissimus and Bunocephalus bicolor, an additional line of polarization was evident in a small area in the anterior part of the macula; therefore, in these forms there was a double bipolar orientation.     

Establishment of hair bundle polarity and orientation in the developing vestibular system of the mouse

The morphological development of the vestibular maculae in the mouse was studied in order to identify elements that may determine how hair-bundle polarity is established. Utricles and saccules develop in parallel. Hair-bundles first appear at embryonic day (E) 13.5. They are initially not polarised and have a kinocilium located at the centre of the cell surface surrounded by stereocilia. Polarisation is rapidly established as the kinocilium becomes eccentrically positioned. The orientation of these polarised bundles is initially not random. It varies systematically across the maculae and the general orientation in utricles is the opposite of that in saccules. At E15.5, in both maculae, hair-bundle orientation angles fall into two populations that differ by approximately 180 degrees defining a line of orientation reversal, the position of which varies little during subsequent maturation. Many more immature hair bundles appear at E15.5 suggesting a second wave of hair cell differentiation is initiated. Otoconial membrane is produced simultaneously across the entire width of both maculae, indicating directional growth of the overlying extracellular matrix is unlikely to influence hair-bundle orientation. Growth of both maculae occurs asymmetrically, essentially outwards from the striola, but it is most pronounced after orientation is defined. Microtubules are prominent in hair cells at the earliest stages of their differentiation, but are oriented parallel to the long axis of the cell and, thus, may not have a role in directing hair-bundle polarity. Microfilament assemblies that are aligned parallel to the apical surface and connect to the adherens junctions in supporting cells could provide a "framework" for hair-bundle orientation. The striated rootlets of ciliary centrioles that are aligned parallel to the cell surface with their tips associated with microfilament assemblies at adherens junctions were the only structural asymmetry identified that might influence the development of hair-bundle polarity.     

Interspecific Variations of Inner Ear Structure in the Deep‐Sea Fish Family Melamphaidae

Inner ear structures are compared among three major genera of the deep‐sea fish family Melamphaidae (bigscales and ridgeheads). Substantial interspecific variation is found in the saccular otoliths, including the presence of a unique otolithic “spur” in the genera Melamphaes and Poromitra. The variation in the saccular otolith is correlated with an increase in the number of hair bundle orientation groups on the sensory epithelia from the genera Scopelogadus to Poromitra to Melamphaes. The diverse structural variations found in the saccule may reflect the evolutionary history of these species. The sensory hair cell bundles in this family have the most variable shapes yet encountered in fish ears. In the saccule, most of the hair bundles are 15–20 μm high, an exceptional height for fish otolithic end organs. These bundles have large numbers of stereovilli, including some that reach the length of the kinocilium. In the utricle, the striolar region separates into two unusually shaped areas that have not been described in any other vertebrates. The brains in all species have a relatively small olfactory bulb and optic tectum, as well as an enlarged posterior cerebellar region that is likely to be involved in inner ear and lateral line (octavolateral) functions. Data from melamphaids support the hypothesis that specialized anatomical structures are found in the ears of some (if not most) deep‐sea fishes, presumably enhancing their hearing sensitivity. Anat Rec, 296:1064–1082, 2013. © 2013 Wiley Periodicals, Inc.     

Hair cell orientation and a possible intraepithelial growth of the macula lagenae in the inner ear of the European eel

The hair cell orientation of the macula lagenae in eels at three different stages of life was examined with the light microscope. Part of the posterior periphery of all three stages was occupied by a few rows of sensory cells having their kinocilium pointing in a direction opposite to the direction of the adjacent sensory cells. The width of this peripheral belt remained the same at all three stages of life, in spite of a considerable growth of the sensory epithelium, and the belt was always confined to the margin. This indicates an intraepithelial growth of at least part of the neuroepithelium of the lagenar macula of the European eel.     

Architecture of the mouse utricle: macular organization and hair bundle heights

Hair bundles are critical to mechanotransduction by vestibular hair cells, but quantitative data are lacking on vestibular bundles in mice or other mammals. Here we quantify bundle heights and their variation with macular locus and hair cell type in adult mouse utricular macula. We also determined that macular organization differs from previous reports. The utricle has approximately 3,600 hair cells, half on each side of the line of polarity reversal (LPR). A band of low hair cell density corresponds to a band of calretinin-positive calyces, i.e., the striola. The relation between the LPR and the striola differs from previous reports in two ways. First, the LPR lies lateral to the striola instead of bisecting it. Second, the LPR follows the striolar trajectory anteriorly, but posteriorly it veers from the edge of the striola to reach the posterior margin of the macula. Consequently, more utricular bundles are oriented mediolaterally than previously supposed. Three hair cell classes are distinguished in calretinin-stained material: type II hair cells, type ID hair cells contacting calretinin-negative (dimorphic) afferents, and type IC hair cells contacting calretinin-positive (calyceal) afferents. They differ significantly on most bundle measures. Type II bundles have short stereocilia. Type IC bundles have kinocilia and stereocilia of similar heights, i.e., KS ratios (ratio of kinocilium to stereocilia heights) approximately 1, unlike other receptor classes. In contrast to these class-specific differences, bundles show little regional variation except that KS ratios are lowest in the striola. These low KS ratios suggest that bundle stiffness is greater in the striola than in the extrastriola.     

Lectin from Griffonia simplicifolia identifies an immature-appearing subpopulation of sensory hair cells in the avian utricle

The sensory hair cells of the inner ear are coated with a variety of glycoproteins and glycolipids which can be identified by the binding of specific lectins. The present study examined the binding patterns of three lectins–Wheat Germ Agglutinin, Peanut Agglutinin, and lectin from Griffonia simplicifolia (Isoform B₄)–in the avian utricle. Each of the lectins exhibited a distinct pattern of hair cell labeling. Wheat Germ Agglutinin (WGA) appeared to label the ciliary bundles of all sensory hair cells. In contrast, the binding of Peanut Agglutinin (PNA) was mainly confined to the ciliary bundles of extrastriolar hair cells. Finally, lectin from Griffonia simplicifolia (GS-IB₄) labeled a subpopulation of hair cells in all regions of the chick utricle. Those bundles were much smaller than the majority of ciliary bundles labeled by either WGA or PNA, and the density of GS-IB₄-labeled bundles in the normal mature utricle was relatively low. Increased densities of GS-IB₄-labeled hair cells were observed in the embryonic utricle and during the process of hair cell regeneration. The observations suggest that GS-IB₄ labels a glycoprotein that is expressed preferentially on the ciliary bundles of immature hair cells.     

Afferent innervation patterns of the saccule in pigeons

The innervation patterns of vestibular saccular afferents were quantitatively investigated in pigeons using biotinylated dextran amine as a neural tracer and three-dimensional computer reconstruction. Type I hair cells were found throughout a large portion of the macula, with the highest density observed in the striola. Type II hair cells were located throughout the macula, with the highest density in the extrastriola. Three classes of afferent innervation patterns were observed, including calyx, dimorph, and bouton units, with 137 afferents being anatomically reconstructed and used for quantitative comparisons. Calyx afferents were located primarily in the striola, innervated a number of type I hair cells, and had small innervation areas. Most calyx afferent terminal fields were oriented parallel to the anterior-posterior axis and the morphological polarization reversal line. Dimorph afferents were located throughout the macula, contained fewer type I hair cells in a calyceal terminal than calyx afferents and had medium sized innervation areas. Bouton afferents were restricted to the extrastriola, with multi-branching fibers and large innervation areas. Most of the dimorph and bouton afferents had innervation fields that were oriented dorso-ventrally but were parallel to the neighboring reversal line. The organizational morphology of the saccule was found to be distinctly different from that of the avian utricle or lagena otolith organs and appears to represent a receptor organ undergoing evolutionary adaptation toward sensing linear motion in terrestrial and aerial species.     

Afferent innervation of the utricular macula in pigeons

Biotinylated dextran amine (BDA) was used to retrogradely label afferents innervating the utricular macula in adult pigeons. The pigeon utriclar macula consists of a large rectangular-shaped neuroepithelium with a dorsally curved anterior edge and an extended medioposterior tail. The macula could be demarcated into several regions based on cytoarchitectural differences. The striola occupied 30% of the macula and contained a large density of type I hair cells with fewer type II hair cells. Medial and lateral extrastriola zones were located outside the striola and contained only type II hair cells. A six- to eight-cell-wide band of type II hair cells existed near the center of the striola. The reversal line marked by the morphological polarization of hair cells coursed throughout the epithelium, near the peripheral margin, and through the center of the type II band. Calyx afferents innervated type I hair cells with calyceal terminals that contained between 2 and 15 receptor cells. Calyx afferents were located only in the striola region, exclusive of the type II band, had small total fiber innervation areas and low innervation densities. Dimorph afferents innervated both type I and type II hair cells with calyceal and bouton terminals and were primarily located in the striola region. Dimorph afferents had smaller calyceal terminals with few type I hair cells, extended fiber branches with bouton terminals and larger innervation areas. Bouton afferents innervated only type II hair cells in the extrastriola and type II band regions. Bouton afferents innervating the type II band had smaller terminal fields with fewer bouton terminals and smaller innervation areas than fibers located in the extrastriolar zones. Bouton afferents had the most bouton terminals on the longest fibers, the largest innervation areas with the highest innervation densities of all afferents. Among all afferents, smaller terminal innervation fields were observed in the striola and large fields were located in the extrastriola. The cellular organization and innervation patterns of the utricular maculae in birds appear to represent an organ in adaptive evolution, different from that observed for amphibians or mammals.     

Zebrafish pax5 regulates development of the utricular macula and vestibular function.

The zebrafish otic vesicle initially forms with only two sensory epithelia, the utricular and saccular maculae, which primarily mediate vestibular and auditory function, respectively. Here, we test the role of pax5, which is preferentially expressed in the utricular macula. Morpholino knockdown of pax5 disrupts vestibular function but not hearing. Neurons of the statoacoustic ganglion (SAG) develop normally. Utricular hair cells appear to form normally but a variable number subsequently undergo apoptosis and are extruded from the otic vesicle. Dendrites of the SAG persist in the utricle but become disorganized after hair cell loss. Hair cells in the saccule develop and survive normally. Otic expression of pax5 requires pax2a and fgf3, mutations in which cause vestibular defects, albeit by distinct mechanisms. Thus, pax5 works in conjunction with fgf3 and pax2a to establish and/or maintain the utricular macula and is essential for vestibular function.     

The distinctive central utricular projections in the herring

The utriculus of the inner ear of clupeid fishes comprises three maculae, each separately innervated. The three nerves were labelled with horseradish peroxidase in the herring and were found to project ipsilaterally to the cerebellum and octavus area and bilaterally to the lateralis zone of the brainstem. The anterior macular nerve terminates dorsally in the octavus area and exclusively in a rostral portion of the anterior octavus nucleus, whereas the middle and posterior macular nerves end ventrally in the octavus area and in the reticular formation. Large middle macular fibers synapse on the Mauthner-cell lateral dendrite.     

Gap junctions between sensory and supporting cells of the utricular and saccular maculae in Anolis carolinensis examined by transmission electron microscopy

Gap junctions, related to projections of sensory cells into supporting cells, occur from supranuclear to basal levels on hair cells, in both saccular and utricular maculae of the lizard, Anolis carolinensis. The larger numbers of junctional projections observed at some levels may indicate a zonular distribution within the neuroepithelia, but only on supracalyceal parts of hair cells in the utricular striola was a series of gap junctions found to be closely associated with the reticular lamina. The dimensions of the junctions, as characterized by the distance between opposite extremities of the arciform membrane appositions, averaged 0.14, 0.19, and 0.28 μm, respectively, on non-calyceal utricular and saccular hair cells, and calyceal cells of the utricular striola. A notable number of the junctional profiles were adjacent or contiguous to open, coated, putatively endocytotic vesicles. These findings are discussed in respect to phylogenetic, developmental, and functional considerations.     

The saccule may be the transducer for directional hearing of nonostariophysine teleosts

The hearing of fishes is transduced by the otolithic end-organs of the eighth nerve. In several nonostariophysine fish, the nerve innervation and hair cell orientation in the saccule, one otolithic organ, suggest that directionality is encoded by a set of mutually perpendicular sensory epithelia. The anterior saccular branch innervates only the hair cell groups oriented along the rostrocaudal body axis which are located at the anterior of the saccule. The posterior saccular branches innervate the hair cell groups oriented along the dorsoventral body axis and are found at the posterior of the saccule.     

Development of the auditory hair cell surface in human fetuses

Summary Thirteen human fetal cochleas were investigated by scanning electron microscopy. Our observations concentrated on the hair cell surface. Ciliogenesis appeared to start during the 11th week of gestation on the inner hair cells (IHCs) and one week later on the outer hair cells (OHCs). The earliest stages of stereociliary development were similar on both types of cell and were characterized by the presence of round bundles of cilia arising from the surrounding microvilli. A three-dimensional V-shaped arrangement suddenly appeared, accompanied by the disappearance of short cilia on the internal side. Between the 20th and the 22nd weeks of gestation, both types of hair cell had an adult stereociliary pattern, i.e. a rectilinear arrangement on IHCs and W-shaped on OHCs. However, there were signs of immaturity, such as a disarray of OHCs and the presence of the kinocilium, suggesting that the surface of the auditory hair cells achieves its maturation during the last trimester of pregnancy.     

The sensory hairs and tectorial membrane of the basilar papilla in the lizardCalotes versicolor

Summary The hearing organ in the lizard, the basilar papilla, is an oblong organ situated in the central opening of the surrounding limbus. The hair cells of the basilar papilla inCalotes versicolor consist of two different types. The type A sensory cells have short hair bundles whose arrangement resembles that of organ pipes, and are situated in the ventral part of the organ. The type B sensory cells have tall, whisk-like hair bundles and are situated in the dorsal part of the basilar papilla. The type A sensory cells are unidirectionally orientated, whereas the type B cells are orientated towards the central sulcus in the papilla. Between the stereocilia, quite close to their base, there is a thin network of interconnecting fibres. Another type of connection is found between the kinocilium and the five adjacent stereocilia. These fibres, however, are situated close to the tips of the relevant cilia. The ventral part of the basilar papilla, i.e., the type A cell population, is covered by a tectorial membrane. Between the microvilli of the supporting cells and the tectorial membrane a network of thin interconnecting filaments is seen. This totally encloses the hair bundles, thus causing them to stand in tubular formations between the sensory epithelium and the tectorial membrane.     

Ultrastructure of the pineal body of the common vampire bat,Desmodus rotundus

The type AB pineal body of the common vampire bat, Desmodus rotundus was recessed and lobulated, was extensively vascularized and intimately related to great veins, and was unassociated with the epithalamic region. The habenular and the posterior commmissures coursed anteriorly and were unassociated with the pineal. The saccular suprapineal recess of the third ventricle extended dorsally juxtaposed to the pineal body. These anatomical features are likely to make pinealectomies in the vampire more difficult to manage. The pineal parenchyma consisted of light pinealocytes surrounded by canaliculi of various sizes, often transmitting unmyelinated nerve fibers and glial processes. Desmosomes were common. The pinealocyte nuclei were large and highly infolded; characteristic cytoplasmic constituents included abundant dilated Golgi complexes associated with clear vesicles, numerous polyribosomes, few single cisternae of ribosome-studded rough endoplasmic reticulum, mitochondria, and occasional multivesicular bodies and lysosomes. Almost all pinealocytes exhibited centrioles and some, in addition, displayed basal bodies but rarely ciliary shafts. A conspicuous feature of the pinealocyte cytoplasm was the presence of branched bundles of intermediate filaments, especially in the perinuclear zone. Siderotic macrophages, lipofuscin-pigment-containing phagocytic cells, mast cells, myelin bodies, and both fenestrated and continuous capillaries were present. The perivascular compartment was densely packed with unmyelinated nerve bundles containing small to large fibers exhibiting axoaxonic densities. Other constituents of the perivascular compartment were club-shaped pinealocyte processes filled with clear vesicles, microtubules, an occasional mitochondrion, glial processes, and collagen fibers. “Synapselike” contacts were observed between the axons and pinealocyte processes. Abundant pinocytotic vesicles in the capillary endothelium indicated active pinocytosis. Myelinated nerve fibers were lacking. The pineal ultrastructure of Desmodus is in part unlike that reported for other mammals, including bats.     

Steady-state stiffness of utricular hair cells depends on macular location and hair bundle structure

Spatial and temporal properties of head movement are encoded by vestibular hair cells in the inner ear. One of the most striking features of these receptors is the orderly structural variation in their mechanoreceptive hair bundles, but the functional significance of this diversity is poorly understood. We tested the hypothesis that hair bundle structure is a significant contributor to hair bundle mechanics by comparing structure and steady-state stiffness of 73 hair bundles at varying locations on the utricular macula. Our first major finding is that stiffness of utricular hair bundles varies systematically with macular locus. Stiffness values are highest in the striola, near the line of hair bundle polarity reversal, and decline exponentially toward the medial extrastriola. Striolar bundles are significantly more stiff than those in medial (median: 8.9 μN/m) and lateral (2.0 μN/m) extrastriolae. Within the striola, bundle stiffness is greatest in zone 2 (106.4 μN/m), a band of type II hair cells, and significantly less in zone 3 (30.6 μN/m), which contains the only type I hair cells in the macula. Bathing bundles in media that break interciliary links produced changes in bundle stiffness with predictable time course and magnitude, suggesting that links were intact in our standard media and contributed normally to bundle stiffness during measurements. Our second major finding is that bundle structure is a significant predictor of steady-state stiffness: the heights of kinocilia and the tallest stereocilia are the most important determinants of bundle stiffness. Our results suggest 1) a functional interpretation of bundle height variability in vertebrate vestibular organs, 2) a role for the striola in detecting onset of head movement, and 3) the hypothesis that differences in bundle stiffness contribute to diversity in afferent response dynamics.