THE ULTRASTRUCTURE OF THE SENSORY HAIRS AND ASSOCIATED ORGANELLES OF THE COCHLEAR INNER HAIR CELL, WITH REFERENCE TO DIRECTIONAL SENSITIVITY

From the apical end of the inner hair cell of the organ of Corti in the guinea pig cochlea protrude four to five rows of stereocilia shaped in a pattern not unlike the wings of a bird. In the area devoid of cuticular substance facing toward the tunnel of Corti lies a consistently present centriole. The ultrastructure of this centriole is similar to that of the basal body of the kinocilium located in the periphery of the sensory hair bundles in the vestibular and lateral line organ sensory cells and to that of the centrioles of other cells. The physiological implications of the anatomical orientation of this centriole are discussed in terms of directional sensitivity.     

Organization of the sensory hairs in the gravity receptors in utricule and saccule of the squirrel monkey

Summary The structural organization of the sensory hairs of the gravity receptors is mainly characterized by the presence of one kinocilium and 40–110 stereocilia on each sensory cell. The spatial arrangement of the kinocilia in relation to the stereocilia presents a polarization, similar to that in the sensory epithelia of the cristae. This polarization, however, is not uniform in the maculae. The direction of polarization varies between groups of several hundred sensory cells. Within one group the sensory cells are all polarized in the same main direction and these groups are considered as functional units.The apparent stiffness and low metabolic activity of the stereocilia suggest their mechanical transmitter function between the otolithic membrane and the sensory cells.The presence of modified kinocilia and basal bodies in other sensory systems raises the question of their significance in sensory receptors. Their unmodified structure in the maculae, however, where the basal bodies are almost identical with centrioles, and the presence of one kinocilium with a basal body and an associated centriole in the supporting cells as well, illustrate their unspecific nature. The centrioles, which later probably become basal bodies, are in close relation to the differentiation of apical cytoplasmic structures such as the kinocilium and the cuticula. This is demonstrated by the appearance of those structures at the bottom of the sensory cell, when the centrioles are situated in this part of the cell.This work was supported by NASA Research Grant NsC 268—62 to the Harvard University Medical School at the Dept. of Otolaryngology, Massachusetts Eye and Ear Infirmary, Boston, Mass, and by U.S.P.H.S. National Institute of Neurological Diseases and Blindness, Grant nos. B 3447 and B. 3779.     

Consequences of Location-Dependent Organ of Corti Micro-Mechanics

The cochlea performs frequency analysis and amplification of sounds. The graded stiffness of the basilar membrane along the cochlear length underlies the frequency-location relationship of the mammalian cochlea. The somatic motility of outer hair cell is central for cochlear amplification. Despite two to three orders of magnitude change in the basilar membrane stiffness, the force capacity of the outer hair cell’s somatic motility, is nearly invariant over the cochlear length. It is puzzling how actuators with a constant force capacity can operate under such a wide stiffness range. We hypothesize that the organ of Corti sets the mechanical conditions so that the outer hair cell’s somatic motility effectively interacts with the media of traveling waves—the basilar membrane and the tectorial membrane. To test this hypothesis, a computational model of the gerbil cochlea was developed that incorporates organ of Corti structural mechanics, cochlear fluid dynamics, and hair cell electro-physiology. The model simulations showed that the micro-mechanical responses of the organ of Corti are different along the cochlear length. For example, the top surface of the organ of Corti vibrated more than the bottom surface at the basal (high frequency) location, but the amplitude ratio was reversed at the apical (low frequency) location. Unlike the basilar membrane stiffness varying by a factor of 1700 along the cochlear length, the stiffness of the organ of Corti complex felt by the outer hair cell remained between 1.5 and 0.4 times the outer hair cell stiffness. The Y-shaped structure in the organ of Corti formed by outer hair cell, Deiters cell and its phalange was the primary determinant of the elastic reactance imposed on the outer hair cells. The stiffness and geometry of the Deiters cell and its phalange affected cochlear amplification differently depending on the location.     

Revisiting planar cell polarity in the inner ear

Since the first implication of the core planar cell polarity (PCP) pathway in stereocilia orientation of sensory hair cells in the mammalian cochlea, much has been written about this subject, in terms of understanding how this pathway can shape the mammalian hair cells and using the inner ear as a model system to understand mammalian PCP signaling. However, many conflicting results have arisen, leading to puzzling questions regarding the actual mechanism and roles of core PCP signaling in mammals and invertebrates. In this review, we summarize our current knowledge on the establishment of PCP during inner ear development and revisit the contrast between wing epithelial cells in Drosophila melanogaster and sensory epithelia in the mammalian cochlea. Notably, we focus on similarities and differences in the asymmetric distribution of core PCP proteins in the context of cell autonomous versus non-autonomous role of PCP signaling in the two systems. Additionally, we address the relationship between the kinocilium position and PCP in cochlear hair cells and increasing results suggest an alternate cell autonomous pathway in regulating PCP in sensory hair cells.     

Kinocilia mediate mechanosensitivity in developing zebrafish hair cells

Mechanosensitive cilia are vital to signaling and development across many species. In sensory hair cells, sound and movement are transduced by apical hair bundles. Each bundle is comprised of a single primary cilium (kinocilium) flanked by multiple rows of actin-filled projections (stereocilia). Extracellular tip links that interconnect stereocilia are thought to gate mechanosensitive channels. In contrast to stereocilia, kinocilia are not critical for hair-cell mechanotransduction. However, by sequentially imaging the structure of hair bundles and mechanosensitivity of individual lateral-line hair cells in?vivo, we uncovered a central role for kinocilia in mechanosensation during development. Our data demonstrate that nascent hair cells require kinocilia and kinocilial links for mechanosensitivity. Although nascent hair bundles have correct planar polarity, the polarity of their responses to mechanical stimuli is initially reversed. Later in development, a switch to correctly polarized mechanosensitivity coincides with the formation of tip links and the onset of tip-link-dependent mechanotransduction.     

Daughter centrioles assemble preferentially towards the nuclear envelope in Drosophila syncytial embryos

Centrosomes are important organizers of microtubules within animal cells. They comprise a pair of centrioles surrounded by the pericentriolar material, which nucleates and organizes the microtubules. To maintain centrosome numbers, centrioles must duplicate once and only once per cell cycle. During S-phase, a single new ‘daughter’ centriole is built orthogonally on one side of each radially symmetric ‘mother’ centriole. Mis-regulation of duplication can result in the simultaneous formation of multiple daughter centrioles around a single mother centriole, leading to centrosome amplification, a hallmark of cancer. It remains unclear how a single duplication site is established. It also remains unknown whether this site is pre-defined or randomly positioned around the mother centriole. Here, we show that within Drosophila syncytial embryos daughter centrioles preferentially assemble on the side of the mother facing the nuclear envelope, to which the centrosomes are closely attached. This positional preference is established early during duplication and remains stable throughout daughter centriole assembly, but is lost in centrosomes forced to lose their connection to the nuclear envelope. This shows that non-centrosomal cues influence centriole duplication and raises the possibility that these external cues could help establish a single duplication site.     

Common themes in centriole and centrosome movements

Centrioles are found in nearly all eukaryotic cells and are required for growth and maintenance of the radial array of microtubules, the mitotic spindle, and cilia and flagella. Different types of microtubule structures are often required at different places in a given cell; centrioles must move around to nucleate these varied structures. Here, we draw together recent data on diverse centriole movements to decipher common themes in how centrioles move. Par proteins establish and maintain the required cellular asymmetry. The actin cytoskeleton facilitates movement of multiple basal bodies. Microtubule forces acting on the cell cortex, and nuclear-cytoskeletal links, are important for positioning individual centrosomes, and during cell division. Knowledge of these common mechanisms can inform the study of centriole movements across biology.     

Developmentally regulated expression of ectonucleotidases NTPDase5 and NTPDase6 and UDP-responsive P2Y receptors in the rat cochlea

Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) regulate complex extracellular P2 receptor signalling pathways in mammalian tissues by hydrolysing extracellular nucleotides to the respective nucleosides. All enzymes from this family (NTPDase1-8) are expressed in the adult rat cochlea. This study reports the changes in expression of NTPDase5 and NTPDase6 in the developing rat cochlea. These two intracellular members of the E-NTPDase family can be released in a soluble form and show preference for nucleoside 5′-diphosphates, such as UDP and GDP. Here, we demonstrate differential spatial and temporal patterns for NTPDase5 and NTPDase6 expression during cochlear development, which are indicative of both cytosolic and extracellular action via pyrimidines. NTPDase5 is noted during the early postnatal period in developing sensory hair cells and supporting Deiters’ cells of the organ of Corti, and primary auditory neurons located in the spiral ganglion. In contrast, NTPDase6 is confined to the embryonic and early postnatal hair cell bundles. NTPDase6 immunolocalisation in the developing cochlea underpins its putative role in hair cell bundle development, probably via cytosolic action, whilst NTPDase5 may have a broader extracellular role in the development of sensory and neural tissues in the rat cochlea. Both NTPDase5 and NTPDase6 colocalize with UDP-preferring P2Y₄, P2Y₆ and P2Y₁₄ receptors during cochlear development, but this strong association was lost in the adult cochlea. Spatiotemporal topographic expression of NTPDase5 and NTPDase6 and P2Y receptors in adult and developing cochlear tissues provide strong support for the role of pyrimidinergic signalling in cochlear development.     

Notch Signaling Limits Supporting Cell Plasticity in the Hair Cell-Damaged Early Postnatal Murine Cochlea

In mammals, auditory hair cells are generated only during embryonic development and loss or damage to hair cells is permanent. However, in non-mammalian vertebrate species, such as birds, neighboring glia-like supporting cells regenerate auditory hair cells by both mitotic and non-mitotic mechanisms. Based on work in intact cochlear tissue, it is thought that Notch signaling might restrict supporting cell plasticity in the mammalian cochlea. However, it is unresolved how Notch signaling functions in the hair cell-damaged cochlea and the molecular and cellular changes induced in supporting cells in response to hair cell trauma are poorly understood. Here we show that gentamicin-induced hair cell loss in early postnatal mouse cochlear tissue induces rapid morphological changes in supporting cells, which facilitate the sealing of gaps left by dying hair cells. Moreover, we provide evidence that Notch signaling is active in the hair cell damaged cochlea and identify Hes1, Hey1, Hey2, HeyL, and Sox2 as targets and potential Notch effectors of this hair cell-independent mechanism of Notch signaling. Using Cre/loxP based labeling system we demonstrate that inhibition of Notch signaling with a γ- secretase inhibitor (GSI) results in the trans-differentiation of supporting cells into hair cell-like cells. Moreover, we show that these hair cell-like cells, generated by supporting cells have molecular, cellular, and basic electrophysiological properties similar to immature hair cells rather than supporting cells. Lastly, we show that the vast majority of these newly generated hair cell-like cells express the outer hair cell specific motor protein prestin.     

The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells

We have generated several stable cell lines expressing GFP-labeled centrin. This fusion protein becomes concentrated in the lumen of both centrioles, making them clearly visible in the living cell. Time-lapse fluorescence microscopy reveals that the centriole pair inherited after mitosis splits during or just after telophase. At this time the mother centriole remains near the cell center while the daughter migrates extensively throughout the cytoplasm. This differential behavior is not related to the presence of a nucleus because it is also observed in enucleated cells. The characteristic motions of the daughter centriole persist in the absence of microtubules (Mts). or actin, but are arrested when both Mts and actin filaments are disrupted. As the centrioles replicate at the G1/S transition the movements exhibited by the original daughter become progressively attenuated, and by the onset of mitosis its behavior is indistinguishable from that of the mother centriole. While both centrioles possess associated gamma-tubulin, and nucleate similar number of Mts in Mt repolymerization experiments. during G1 and S only the mother centriole is located at the focus of the Mt array. A model, based on differences in Mt anchoring and release by the mother and daughter centrioles, is proposed to explain these results.     

Preferential expression of transient potassium current (IA) by 'short' hair cells of the chick's cochlea

We have made a comparative study of the membrane properties of tall and short hair cells isolated from a selected region of the chick's cochlea. Tall hair cells are analogous to inner cochlear hair cells of mammals, and like those, are presynaptic to the majority of afferent neurons in the cochlea. Short hair cells, like mammalian outer hair cells, are the postsynaptic targets of efferent neurons that inhibit the cochlea. Voltage-clamp recordings have revealed that short hair cells have an inactivating potassium (K) current, IA, whereas tall hair cells have little or none. Short hair cells are also sensitive to the cholinergic agonist carbachol, whereas tall hair cells are not. This pattern is in accord with the selective distribution of efferent cholinergic synapses in the cochlea. Although IA is completely inactivated at the resting potential of the short hair cells, cholinergic agonists can hyperpolarize these cells by as much as 30 mV. This hyperpolarization removes inactivation and allows IA to modulate subsequent voltage-dependent processes in short hair cells. It is concluded that IA could increase the high frequency response of the hair cell by decreasing membrane resistance and thus the membrane time constant after inhibition. This will be of particular importance to cochlear function if short hair cells produce voltage-dependent movements, as do mammalian outer hair cells.     

Regulation of cochlear convergent extension by the vertebrate planar cell polarity pathway is dependent on p120-catenin

The vertebrate planar cell polarity (PCP) pathway consists of conserved PCP and ciliary genes. During development, the PCP pathway regulates convergent extension (CE) and uniform orientation of sensory hair cells in the cochlea. It is not clear how these diverse morphogenetic processes are regulated by a common set of PCP genes. Here, we show that cellular contacts and geometry change drastically and that the dynamic expression of N-cadherin and E-cadherin demarcates sharp boundaries during cochlear extension. The conditional knockout of a component of the adherens junctions, p120-catenin, leads to the reduction of E-cadherin and N-cadherin and to characteristic cochlear CE defects but not misorientation of hair cells. The specific CE defects in p120-catenin mutants are in contrast to associated CE and hair cell misorientation defects observed in common PCP gene mutants. Moreover, the loss-of-function of a conserved PCP gene, Vangl2, alters the dynamic distribution of N-cadherin and E-cadherin in the cochlea and causes similar abnormalities in cellular morphology to those found in p120-catenin mutants. Conversely, we found that Pcdh15 interacts genetically with PCP genes to regulate the formation of polar hair bundles, but not CE defects in the cochlea. Together, these results indicate that the vertebrate PCP pathway regulates CE and hair cell polarity independently and that a p120-catenin-dependent mechanism regulates CE of the cochlea.     

Structure and function of the Nautilus statocyst.

The two equilibrium receptor organs (statocysts) of Nautilus are avoid sacks, half-filled with numerous small, free-moving statoconia and half with endolymph. The inner surface of each statocyst is lined with 130,000-150,000 primary sensory hair cells. The hair cells are of two morphological types. Type A hair cells carry 10-15 kinocilia arranged in a single ciliary row; they are present in the ventral half of the statocyst. Type B hair cells carry 8-10 irregularly arranged kinocilia; they are present in the dorsal half of the statocyst. Both type of hair cells are morphologically polarized. To test whether these features allow the Nautilus statocyst to sense angular accelerations, behavioural experiments were performed to measure statocyst-dependent funnel movements during sinusoidal oscillations of restrained Nautilus around a vertical body axis. Such dynamic rotatory stimulation caused horizontal phase-locked movements of the funnel. The funnel movements were either in the same direction (compensatory funnel response), or in the opposite direction (funnel follow response) to that of the applied rotation. Compensatory funnel movements were also seen during optokinetic stimulation (with a black and white stripe pattern) and during stimulations in which optokinetic and statocyst stimulations were combined. These morphological and behavioural findings show that the statocysts of Nautilus, in addition to their function as gravity receptor organs, are able to detect rotatory movements (angular accelerations) without the specialized receptor systems (crista/cupula systems) that are found in the statocysts of coleoid cephalopods. The findings further indicate that both statocyst and visual inputs control compensatory funnel movements.     

Centriole is the pivot co-ordinating dynamic signaling for cell proliferation and organization during early development in the vertebrates

Vertebrates have an elaborate and functionally segmented body. It evolves from a single cell by systematic cell proliferation but attains a complex body structure with exquisite precision. This development requires two cellular events: cell cycle and ciliogenesis. For these events, the dynamic molecular signaling is converged at the centriole. The cell cycle helps in cell proliferation and growth of the body and is a highly regulated and integrated process. Its errors cause malignancies and developmental disorders. The cells newly proliferated are organized during organogenesis. For a cellular organization, dedicated signaling hubs are developed in the cells, and most often cilia are utilized. The cilium is generated from one of the centrioles involved in cell proliferation. The developmental signaling pathways hosted in cilia are essential for the elaboration of the body plan. The cilium's compartmental seclusion is ideal for noise-free molecular signaling and is essential for the precision of the body layout. The dysfunctional centrioles and primary cilia distort the development of body layout that manifest as serious developmental disorders. Thus, centriole has a dual role in the growth and cellular organization. It organizes dynamically expressed molecules of cell cycle and ciliogenesis and plays a balancing act to generate new cells and organize them during development. A putative master molecule may regulate and co-ordinate the dynamic gene expression at the centrioles. The convergence of many critical signaling components at the centriole reiterates the idea that centriole is a major molecular workstation involved in elaborating the structural design and complexity in vertebrates.     

Control of daughter centriole formation by the pericentriolar material

Controlling the number of its centrioles is vital for the cell, as supernumerary centrioles cause multipolar mitosis and genomic instability. Normally, one daughter centriole forms on each mature (mother) centriole; however, a mother centriole can produce multiple daughters within a single cell cycle. The mechanisms that prevent centriole 'overduplication' are poorly understood. Here we use laser microsurgery to test the hypothesis that attachment of the daughter centriole to the wall of the mother inhibits formation of additional daughters. We show that physical removal of the daughter induces reduplication of the mother in S-phase-arrested cells. Under conditions when multiple daughters form simultaneously on a single mother, all of these daughters must be removed to induce reduplication. The number of daughter centrioles that form during reduplication does not always match the number of ablated daughter centrioles. We also find that exaggeration of the pericentriolar material (PCM) by overexpression of the PCM protein pericentrin in S-phase-arrested CHO cells induces formation of numerous daughter centrioles. We propose that that the size of the PCM cloud associated with the mother centriole restricts the number of daughters that can form simultaneously.     

GIANT CENTRIOLE FORMATION IN SCIARA

Although somatic tissues of Sciara contain 9-membered centrioles, germ line tissues develop giant centrioles with 60–90 singlet tubules disposed in an oval array. Some 9-membered centrioles still may be seen in second instar spermatogonia. Each of these centrioles is associated with a larger "daughter" or secondary centriole at right angles to it. Most centrioles of second instar spermatogonia consist of 20–50 singlet tubules arranged in an oval, sometimes associated with an even larger secondary centriole. The more recently formed centriole of a pair is distinguishable from its partner by a concentric band of electron-opaque material inside its tubules. If a pair of centrioles at right angles to each other is pictured as a "T" formed by two cylinders, the secondary centriole is always the stem of the T; the primary centriole is the top. The two centrioles are oriented at the pole of the mitotic spindle so that the tubules of the primary centriole are parallel to the spindle axis. Each daughter cell receives a pair of centrioles and, during interphase, each of these centrioles gives rise to a new daughter centriole. A Golgi area of characteristic morphology is found in association with centrioles shortly after two new ones have formed. We conclude that in Sciara a centriole may give rise to a daughter morphologically different from itself. Whether the daughter is a 9-membered or giant centriole depends on the tissue type and stage of development.     

Centrioles in the cell cycle. I. Epithelial cells

A study was made of the structure of the centrosome in the cell cycle in a nonsynchronous culture of pig kidney embryo (PE) cells. In the spindle pole of the metaphase cell there are two mutually perpendicular centrioles (mother and daughter) which differ in their ultrastructure. An electron-dense halo, which surrounds only the mother centriole and is the site where spindle microtubules converge, disappears at the end of telophase. In metaphase and anaphase, the mother centriole is situated perpendicular to the spindle axis. At the beginning of the G1 period, pericentriolar satellites are formed on the mother centriole with microtubules attached to them; the two centrioles diverge. The structures of the two centrioles differ throughout interphase; the mother centriole has appendages, the daughter does not. Replication of the centrioles occurs approximately in the middle of the S period. The structure of the procentrioles differs sharply from that of the mature centriole. Elongation of procentrioles is completed in prometaphase, and their structure undergoes a number of successive changes. In the G2 period, pericentriolar satellites disappear and some time later a fibrillar halo is formed on both mother centrioles, i.e., spindle poles begin to form. In the cells that have left the mitotic cycle (G0 period), replication of centrioles does not take place; in many cells, a cilium is formed on the mother centriole. In a small number of cells a cilium is formed in the S and G2 periods, but unlike the cilium in the G0 period it does not reach the surface of the cell. In all cases, it locates on the centriole with appendages. At the beginning of the G1 period, during the G2 period, and in nonciliated cells in the G0 period, one of the centrioles is situated perpendicular to the substrate. On the whole, it takes a mature centriole a cycle and a half to form in PE cells.     

Regeneration of Stereocilia of Hair Cells by Forced Atoh1 Expression in the Adult Mammalian Cochlea

The hallmark of mechanosensory hair cells is the stereocilia, where mechanical stimuli are converted into electrical signals. These delicate stereocilia are susceptible to acoustic trauma and ototoxic drugs. While hair cells in lower vertebrates and the mammalian vestibular system can spontaneously regenerate lost stereocilia, mammalian cochlear hair cells no longer retain this capability. We explored the possibility of regenerating stereocilia in the noise-deafened guinea pig cochlea by cochlear inoculation of a viral vector carrying Atoh1, a gene critical for hair cell differentiation. Exposure to simulated gunfire resulted in a 60–70 dB hearing loss and extensive damage and loss of stereocilia bundles of both inner and outer hair cells along the entire cochlear length. However, most injured hair cells remained in the organ of Corti for up to 10 days after the trauma. A viral vector carrying an EGFP-labeled Atoh1 gene was inoculated into the cochlea through the round window on the seventh day after noise exposure. Auditory brainstem response measured one month after inoculation showed that hearing thresholds were substantially improved. Scanning electron microscopy revealed that the damaged/lost stereocilia bundles were repaired or regenerated after Atoh1 treatment, suggesting that Atoh1 was able to induce repair/regeneration of the damaged or lost stereocilia. Therefore, our studies revealed a new role of Atoh1 as a gene critical for promoting repair/regeneration of stereocilia and maintaining injured hair cells in the adult mammal cochlea. Atoh1-based gene therapy, therefore, has the potential to treat noise-induced hearing loss if the treatment is carried out before hair cells die.     

Retinoic acid signaling is necessary for the development of the organ of Corti.

The cellular mosaic of the mammalian organ of Corti represents one of the most highly ordered structures in any vertebrate system. A single row of inner hair cells and three or four rows of outer hair cells extend along the basal-to-apical axis of the cochlea. The factors that play a role in the development of specific cell types within the cochlea are largely unknown; however, the results of previous studies have strongly suggested that retinoic acid plays a role in the development of cells as hair cells. To determine whether cochlear progenitor cells can respond directly to retinoic acid, the expression patterns for each of the RAR and RXR receptors within the embryonic cochlear duct were determined by in situ hybridization. Results indicate that RARalpha, RXRalpha, and RXRgamma are initially expressed throughout the cochlear duct. As development continues, the expression of each receptor becomes more intense in cells that will develop as hair cells. At the same time, receptor expression is down-regulated in cells that will develop as nonsensory cell types. To determine the effects of retinoic acid signaling during the development of the organ of Corti, activation of retinoid receptors was blocked in cultures of the embryonic cochlea through receptor-specific antagonism or inhibition of retinoic acid synthesis. Results indicate that inhibition of retinoic acid signaling induces a significant decrease in the number of cells that develop as hair cells and a disruption in the development of the organ of Corti. These results demonstrate that cells within the developing cochlea can respond to retinoic acid and that signaling by retinoic acid is necessary for the normal development of the organ of Corti.     

Comparative light and electron microscopic study of the auditory organs of two species of fishes (pisces): Hyphessobrycon simulans (Ostariophysi) and Poecilia reticulata (Acanthopterygii).

Hyphessobrycon simulans has a Weberian apparatus for transmission of sound energy to the auditory organ, whereas Poecilia reticulata does not. The fine structure of the auditory organs is identical in the two species. The better hearing - expressed by large bandwidth and high sensitivity - typical of the Ostariophysi - seems to be based exclusively on the presence of the Weberian apparatus. The sensory epithelium of the saccule and the lagena is made up of hair (sensory) cells and supporting cells. The vertically orientated macula sacculi is divided into a dorsal and a ventral cell area with oppositely arranged hair-cell kinocilia. The sagitta takes up the center of the saccule and shows only three small sites with connections to the otolithic membrane. Remarkably, the dorsal sensory cells are connected to the ventral part of the otolith, but the ventral cells are connected to the dorsal part. The macula of the lagena also comprises a dorsal and a ventral cell area with oppositely arranged hair cells. The sensory cells in all maculae are of type II. They exhibit a striking apical cell protrusion, the cuticular villus. It is partially fused with the kinocilium in the contact zones and joined to the otolithic membrane. The cuticular villus probably stabilizes the long kinocilia.