Further scanning electron microscope studies of lizard auditory papillae

The papillae basilares of 12 species of lizards from seven different families were studied by SEM. The iguanids, Sceloporus magister and S. occidentalis, have typical “iguanid type” papillae with central short-ciliated unidirectional hair cell segments and apical and basal long-ciliated bidirectional hair cell segments. These species of Sceloporus are unique among iguanids in that the bidirectional segments consist of but two rows of hair cells. The agamids, Agama agama and Calotes nigrolabius, have an “agamid-anguid type” papilla consisting of an apical short-ciliated unidirectional hair cell segment and a longer basal bidirectional segment. Agama agama is unusual in having a few long-ciliated hair cells at the apical end of the apical short-ciliated segment. The agamid, Uromastix sp., has an “iguanid type” papilla with a central short-ciliated unidirectional segment and apical and basal bidirectional segments. The anguid, Ophisaurus ventralis, has an “iguanid” papillar pattern with the short-ciliated segment centrally located. All the short-ciliated hair cells of the above species are covered by a limbus-attached tectorial network or cap and the long-ciliated hair cells, only by loose tectorial strands. The lacertids, Lacerta viridis and L. galloti, have papillae divided into two separate segments. The shorter apical segment consists of opposingly oriented, widely separated short-ciliated cells covered by a heavy tectorial membrane. The apical portion of the longer basal segment consists of unidirectionally oriented hair cells, while the greater part of the segment has opposingly oriented hair cells. The xantusiids, Xantusia vigilis and X. henshawi, have papillae made up of separate small apical segments and elongated basal segments. The apical hair cells are largely, but not exclusively, unidirectional and are covered by a heavy tectorial cap. The basal strip is bidirectional and the hair cells are covered by sallets. The kinocilial heads are arrowhead-shaped. The papilla of the cordylid, Cordylus jonesii, is very similar to that of Xantusia except that the apical segment is not completely separated from the basal strip. The papilla of the Varanus bengalensis is divided into a shorter apical and a longer basal segment. The hair cells of the entire apical and the basal three quarters of the basal segment are opposingly oriented, not with reference to the midpapillary axis but randomly to either the neural or abneural direction. The apical quarter of the basal segment contains unidirectional, abneurally oriented hair cells. The entire papilla is covered by a dense tectorial membrane. The functional correlations of the above structural variables are discussed.     

Scanning electron microscope studies of the papilla basilaris of some turtles and snakes

The papillae basilares of three species of turtles and four species of snakes were studied by SEM. The papillae of turtle are relatively large among reptiles and are characterized by a long, horizontal middle section resting on wide basilar membrane. Both terminal ends of the papilla extend onto the surrounding limbus in the form of a forked or "T" -shaped end or as a curved, "hook"- like processes. Details vary with the species. In the three species of turtles studied, there were between 1,100 and 1,400 hair cells on a papilla. The tectorial membrane covering the horizontal portion of the papilla is heavy in appearance and tightly attached to the kinocilial bulbs. The terminal ends of the papilla are covered by a thin gelatinous material. In addition, mat-like tectorial network covers the supporting cells and extends from the microvilli of the supporting cells to the overlying tectorial membrane. All hair cells are unidirectionally and abneurally oriented. The supporting cell surfaces form a large part of the papilla and, thus, hair cell density is low. The papillae of the two boid snake species studied are moderately long among snakes and contain a moderate number of hair cells (574 in Epicrates and 710-780 in Constrictor). Papillar form is elongate, avoid, or canoe-shaped. The tectorial membrane may be either highly fenestrated or moderately dense and covers all but a few of the terminal hair cells. A tectorial-like mat covers all but a few of the terminal hair cells. Most hair cells are unidirectionally and abneurally oriented. A few terminal cells in boids may show reverse orientation. Hair cell density is similar to that of turtles.     

A scanning electron microscope study of the papilla basilaris of Gekko gecko

Summary The sensory hair cells of the ventral 2/3 of the papilla basilaris of Gekko gecko are divided into anterior (pre-axial) and posterior (post-axial) portions by a mid-axial gap or hiatus where there are no hair cells. There is no separation of the hair cells in the dorsal third of the papilla. There are three tectorial membrane modifications: an attached thickened membrane covering the pre-axial hair cells, sallets covering the post-axial hair cells, and an attached filamentous membrane covering the dorsal hair cells. The number of hair cells is greatest ventrally and decreases dorsally. There are approximately 2000 to 2100 hair cells. The kinocilia of the hair cells of the anterior halves of both the pre- and the post-axial vertical hair-cell rows are oriented posteriorly, while the kinocilia of the posterior halves are oriented anteriorly. The kinocilia of the hair cells of the dorsal third of the papilla are mostly oriented posteriorly. Thus, kinocilial orientation of the ventral 2/3 of the papilla is doubly bidirectional, and the dorsal 1/3, largely unidirectional.I would like to thank Ms. Maria Maglio for her skill in handling the technical aspects of the scanning electron microscopy as well as her artistry in achieving photographic excellence on the scope, David Akers for expert photographic assistance, and Wayne Emery for the drawings. Research sponsored by United States Public Health Service Grant NS-09231.     

The cellular organization and nervous supply of the basilar papilla in the lizard,Calotes versicolor

Summary The basilar papilla of the lizard Calotes versicolor contains about 225 sensory cells. These are of two types: the short-haired type A cells in the ventral (apical) part of the organ, and the type B cells with long hair bundles, in the dorsal (basal) part of the organ. The type A cells are unidirectionally oriented and are covered by a tectorial membrane while the type B cells lack a covering structure and their hair bundles are oriented bidirectionally. Apart from those differences, the type A and type B cells are similar. They are columnar, and display the features common to most sensory cells in inner ear epithelia. The sensory cells are separated by supporting cells, which have long slender processes that keep the sensory cells apart. Close to the surface of the basilar papilla a terminal bar of specialized junctions interlocks adjacent cells. Below this, adjacent supporting cells are linked by an occluding junction.The cochlear nerve enters from the medial (neural) aspect. The fibres of the nerve lose their myelin sheaths as they enter the basilar papilla. Each sensory cell is associated with several nerve endings. All the nerves identified were afferent. Marked variations were seen between nerve endings in the basilar papilla, but no morphological equivalents of any functional differences were observed.This work is supported by grant no. B76-12X-00720-11A from the Swedish Medical Research Council, and by funds from the Karolinska Institute, Stockholm, Sweden.     

The organization and isolating function of insect rectal sheath cells: a freeze-fracture study.

In termites and roaches the well-defined rectal papillae each comprise a layer of columnar principal cells specialized for active transport and a layer of basal cells. The whole cell group is entirely surrounded by several series of flattened 'sheath cells' (formerly termed 'junctional cells') which abut onto the basal components of the papilla. The sheath cells secrete a specialized sclerified cuticle which forms the framework of the papilla. Their regularly pleated apical membrane is closely apposed to the cuticle and contains parallel and closely spaced rows of intramembranous particles. at this level, no subcuticular space is present and hence the space associated with the apical surface of the principal cells is defined as an isolated compartment. Typical septate junctions are present between the sheath and basal cells; however those linking adjacent sheath cells are structurally unusual: they extend to the basal surface rather than being restricted to the apical zone, are frequently interrupted and in replicas are represented by relatively short and irregularly oriented particle rows. Moreover, lateral sheath cell contacts display two further peculiarities: absence of an apical desmosomal ring and paucity of gap junctions. Structural observations suggest that the sheath cells isolate the principal cells from communication with the hemolymph, consequently enhancing their efficiency in water and ionic regulation. Comparable cells have been described in a number of insects, but the 'isolation' system presents varying degrees of complexity, for which an evolutionary scheme is proposed.     

The tectorial membrane of the lizard ear: types of structure

This study is concerned with the forms of the tectorial membrane in the lizard ear and its manner of attachment to the ciliary tufts of the hair cells. These structures and their variations were observed in 20 species representing eight families of lizards. Three forms of tectorial membrane were found, a continuous form that extends throughout the length of the auditory papilla, an abbreviated form that reaches the papilla only in one region, and a dendritic form that is particularly narrow at first and then branches extensively to supply all the hair cells. Occasionally the lower edge of the tectorial membrane makes direct connections with the hair tufts. More often there are special connecting structures between the membrane and the hair tufts. Seven types of these structures were identified, as follows: (1) simple fibers, (2) open network, (3) heavy network, (4) fiber plate, (5) finger processes, (6) sallets, and (7) remote connections. These types of tectorial connections are described and illustrated.     

Ultrastructure of free neuromasts of Bathygobius fuscus (gobiidae) and canal neuromasts of Apogon cyanosoma (apogonidae)

Light and electron microscopic observations were made on the lateral line organs of the free neuromasts of the goby Bathygobius fuscus and the canal neuromasts of the cardinal fish Apogon cyanosoma. As in other lateral line systems, each neuromast consists of hair cells, supporting cells and mantle supporting cells, the whole being covered by a cupula. In B. fuscus the free neuromasts are mounted on papillae and have hair cells with stereocilia up to 2.5 μm long and a single kinocilium at least 25 μm long. Each neuromast is covered by a vane-like cupula that can be divided into two regions. The central region over the sensory area contains columns of myelin-like figures. These figures are absent from the outer region covering the mantle. The canal neuromasts of A. cyanosoma are diamond-shaped with up to 1,500 hair cells. The cupula is unusual in having a channel that lies over the sensory region. The hair cells have up to 45 stereocilia, the tallest reaching 2.5 μm, and a kinocilium at least 5 μm long. Tip links are shown for the first time between rows of stereocilia of the hair cells of lateral line neuromasts. The presence of tip links has now been demonstrated for all acousticolateral hair cell systems.     

The lizard ear: Gekkonidae

The gecko ear was studied in 36 species belonging to 24 genera. This receptor has attained an advanced level of structure and performance in this group of lizards, but there are many variations among species. To a large extent these variations follow subfamily lines as represented in Kluge's system of classification. Brief consideration is given to features of the outer and middle ear, but chief concern is with inner ear structures and their relations to auditory sensitivity as represented by the cochlear potentials. The auditory papilla is segmented, with a dorsal portion whose hair cells have their ciliary tufts attached to a tectorial membrane, and a ventral portion in which these cells form tow assemblages, one with tectorial connections and the other with connections to a line of sallets. The dorsal segment varies greatly in length and in the form of ciliary orientation. In Eublepharinae and most Gekkoninae the ciliary orientation is unidirectional, and the degree of sensitivity relates to the length of this segment. In Diplodactylinae and Sphaerodactylinae the orientation is bidirectional, and this segment functionally hardly differs from the ventral segment. Auditory sensitivity as measured in terms of the cochlear potentials shows close relations with subfamily groupings, except for the Gekkoninae in which considerable diversity is found. The evidence from structural differentiation, along with that derived from the forms of the cochlear potential functions, leads to the suggestion that these ears possess a high degree of pitch discrimination and capability for the analysis of complex sounds.     

Morphological and fine structural features of the basilar papilla in ambystomatid salamanders (Amphibia; Caudata)

The morphology and fine structure of the basilar recess and basilar papilla were investigated in four species of salamanders from the family Ambystomatidae. The otic relationships of the recess and papilla to the proximal part of the lagena and saccule are described, and new terminology is suggested for the periotic relationships of the basilar recess to a diverticulum of an intracapsular periotic sac. The basilar papilla consists of supporting cells united laterally by gap junctions, capped by microvilli uniformly arranged around a short, central cilium, and hair cells that typically show several synapses with a single afferent nerve fiber, each marked by a rounded synaptic body surrounded by vesicles. In contrast to anuran basilar papillae, efferent nerve terminals were observed in synapse with hair cells and, rarely, upon afferent fibers. The distal half of the ambystomatid papilla contained hair cells capped by tall ciliary bundles, with kinocilia that show swellings near their tips with delicate attachments to adjacent tall stereocilia. A tectorial body covers only this region of the papilla. Hair cells with shorter stereocilia, situated in the proximal half and at the papillar margins, are related only to filamentous extensions of the tectorial body. The ambystomatid basilar recess and papilla are compared to auditory end-organs in other vertebrates, and it is suggested that a basic distinction can be made between aural neuroepithelia in amniotes versus that in nonamniotic vertebrate ears.     

Molecular cloning of chick beta-tectorin, an extracellular matrix molecule of the inner ear

The tectorial membrane is an extracellular matrix lying over the apical surface of the auditory epithelium. Immunofluorescence studies have suggested that some proteins of the avian tectorial membrane, the tectorins, may be unique to the inner ear (Killick, R., C. Malenczak, and G. P. Richardson. 1992. Hearing Res. 64:21-38). The cDNA and deduced amino acid sequences for chick beta-tectorin are presented. The cDNA encodes a protein of 36,902.6 D with a putative signal sequence, four potential N-glycosylation sites, 13 cysteines, and a hydrophobic COOH terminus. Western blots of two-dimensional gels using antibodies to a synthetic peptide confirm the identity of the cDNA. Southern and Northern analysis suggests that beta-tectorin is a single-copy gene only expressed in the inner ear. The predicted COOH terminus is similar to that of glycosylphosphatidylinositol-linked proteins, and antisera raised to this region react with in vitro translation products of the cDNA clone but not with mature beta-tectorin. These data suggest beta- tectorin is synthesized as a glycosylphosphatidyl-inositol-linked precursor, targeted to the apical surface of the sensory epithelium by the lipid moiety, and then further processed. Sequence analysis indicates the predicted protein possesses a zona pellucida domain, a sequence that is common to a limited number of other matrix-forming proteins and may be involved in the formation of filaments. In the cochlear duct, beta-tectorin is expressed in the basilar papilla, in the clear cells and the cuboidal cells, as well as in the striolar region of the lagena macula. The expression of beta-tectorin is associated with hair cells that have an apical cell surface specialization known as the 275-kD hair cell antigen restricted to the basal region of the hair bundle, suggesting that matrices containing beta-tectorin are required to drive this hair cell type.     

Peripheral auditory processing in the bobtail lizard Tiliqua rugosa

Summary We have labelled single physiologically-characterized primary auditory neurones in the bobtail lizard and traced them to their innervation sites within the basilar papilla. The distribution of stained fibre terminals shows that low frequencies (up to a characteristic frequency, CF, of about 0.8 kHz) are processed in the smaller apical segment of the papilla and medium to high frequencies in the much longer basal segment. It is possible that the frequency ranges of these segments partly overlap in individual animals.The tonotopic organization of the basal segment is well described by an exponential relationship; the CF increases towards the basal end. Systematic, peripheral recordings from the auditory nerve very close to the papilla confirm this tonotopicity for the basal segment.The apical segment of the papilla shows an unusual tonotopic organization in that the CF appears to increase across the epithelium, from abneural to neural. A tonotopicity in this direction has not previously been demonstrated in vertebrates.All stained neurones branched within the basilar papilla to innervate, typically, between 4 and 14 hair cells. The branching patterns of fibres innervating in the apical and basal papillar segment, respectively, show characteristic differences. Apical fibres tend to innervate hair cells with the same morphological polarity and often branch extensively along the segment. Basal fibres, in contrast, typically innervate about equal numbers of hair cells of opposing polarity and are more restricted in their longitudinal branching.Abbreviation CF characteristic frequency     

The midline (notochord and notoplate) patterns the cell motility underlying convergence and extension of the Xenopus neural plate

We investigated the role of the dorsal midline structures, the notochord and notoplate, in patterning the cell motilities that underlie convergent extension of the Xenopus neural plate. In explants of deep neural plate with underlying dorsal mesoderm, lateral neural plate cells show a monopolar, medially directed protrusive activity. In contrast, neural plate explants lacking the underlying dorsal mesoderm show a bipolar, mediolaterally directed protrusive activity. Here, we report that "midlineless" explants consisting of the deep neural plate and underlying somitic mesoderm, but lacking a midline, show bipolar, mediolaterally oriented protrusive activity. Adding an ectopic midline to the lateral edge of these explants restores the monopolar protrusive activity over the entire extent of the midlineless explant. Monopolarized cells near the ectopic midline orient toward it, whereas those located near the original, removed midline orient toward this midline. This behavior can be explained by two signals emanating from the midline. We postulate that one signal polarizes neural plate deep cells and is labile and short-lived and that the second signal orients any polarized cells toward the midline and is persistent.     

Cell surface expansion in polarly growing root hairs of Medicago truncatula

Fluorescent microspheres were used as material markers to investigate the relative rates of cell surface expansion at the growing tips of Medicago truncatula root hairs. From the analysis of tip shape and microsphere movements, we propose three characteristic zones of expansion in growing root hairs. The center of the apical dome is an area of 1- to 2- microm diameter with relatively constant curvature and high growth rate. Distal to the apex is a more rapidly expanding region 1 to 2 microm in width exhibiting constant surges of off-axis growth. This middle region forms an annulus of maximum growth rate and is visible as an area of accentuated curvature in the tip profile. The remainder of the apical dome is characterized by strong radial expansion anisotropy where the meridional rate of expansion falls below the radial expansion rate. Data also suggest possible meridional contraction at the juncture between the apical dome and the cell body. The cell cylinder distal to the tip expands slightly over time, but only around the circumference. These data for surface expansion in the legume root hair provide new insight into the mechanism of tip growth and the morphogenesis of the root hair.     

The lizard ear: Cordylus, Platysaurus and Gerrhosaurus

In further consideration of the lizard ear, the fine structure of the cochlea has been investigated and related to auditory sensitivity in members of the family Cordylidae. The ear of this group of lizards is unusual in that a tectorial membrane is present only in a modified and seemingly vestigial form, and this membrane makes no connections with the auditory hair cells. These cells are provided instead with a series of sallets, small bodies extending in a single row through the dorsal and middle regions of the cochlea, where they rest upon the tips of the ciliary tufts and evidently bring about a stimulation of the hair cells because of their inertia. At the ventral end of the cochlea this line of sallets ends, and here is a single, relatively enormous structure, the culmen papillae, that serves a similar purpose for a large group of hair cells. Consideration is given to the manner of stimulation of the auditory sense cells in these species in relation to others with the usual arrangements involving connections between the ciliary tufts and a tectorial membrane. Included also is a study of a species of Gerrhosaurus, which some have included in the cordylid family and others have placed in a family of its own. The cochlear structure in this species is similar to that of the cordylids in many respects but differs in the ventral region, where instead of the culmen there is a heavy tectorial plate, similarly covering a large number of hair cells but connected to a tectorial membrane. The functioning of these ears is assessed in terms of the cochlear potentials, and is found to vary with species from better than average to excellent in comparison with other lizards investigated. The structural differentiation also is of fairly high degree, and hence it appears that ears without tectorial connections, or with such connections only in a limited region of the cochlea, can perform in a highly serviceable manner.     

Simulation of the response of the inner hair cell stereocilia bundle to an acoustical stimulus

Mammalian hearing relies on a cochlear hydrodynamic sensor embodied in the inner hair cell stereocilia bundle. It is presumed that acoustical stimuli induce a fluid shear-driven motion between the tectorial membrane and the reticular lamina to deflect the bundle. It is hypothesized that ion channels are opened by molecular gates that sense tension in tip-links, which connect adjacent stepped rows of stereocilia. Yet almost nothing is known about how the fluid and bundle interact. Here we show using our microfluidics model how each row of stereocilia and their associated tip links and gates move in response to an acoustical input that induces an orbital motion of the reticular lamina. The model confirms the crucial role of the positioning of the tectorial membrane in hearing, and explains how this membrane amplifies and synchronizes the timing of peak tension in the tip links. Both stereocilia rotation and length change are needed for synchronization of peak tip link tension. Stereocilia length change occurs in response to accelerations perpendicular to the oscillatory fluid shear flow. Simulations indicate that nanovortices form between rows to facilitate diffusion of ions into channels, showing how nature has devised a way to solve the diffusive mixing problem that persists in engineered microfluidic devices.     

Morphology, and muscle- and papilla-volume ratios, of the tongue of Laudakia stellio (Agamidae, Squamata): a histological and stereological study

We examined the histological structure of the tongue of Laudakia stellio, the starred agama lizard (Agamidae, Squamata), under light microscopy. We also investigated the muscle and papilla volume ratios, with volumes of each aspect of interest estimated according to the Cavalieri method. The macroscopically short, thick and muscle-rich front tip of the tongue of L. stellio does not show any bifurcation, and under light microscopy, the oval-shaped papilla-free front tip was seen to be covered by keratinized stratified epithelium. The dorsal and ventral parts were different, with the former partially covered by keratinized stratified epithelium and rich in secretory glands and secretory cells. The ventral part, which contained keratinized stratified cells, had a flat surface with no papillae. The dorsal surface of the anterior and posterior parts contained fungiform papillae, with the apical parts of these papillae containing minimal keratin; the interpapillar space was covered by keratin-free squamous stratified epithelium. The middle section of the tongue contained cylindrical-type papillae, with serous and mucous secretory glands and ducts at their base. Finally, the frontal and middle parts of the ventral and dorsal surfaces did not contain any taste buds, although there were some in the hind part of the dorsal surface. As morphometric estimates of volumes of the muscles and papillae, the mean volume ratios (relative to total tongue volume)+/-standard deviation were 0.66+/-0.03 and 0.33+/-0.03, with mean coefficients of error of 0.02 and 0.03, respectively.     

Diversity of form in the amphibian papilla of Puerto Rican frogs

Summary In modern frogs, the amphibian papilla exhibits a caudal extension whose shape, relative length, and proportion of hair cells vary markedly from species to species. Tuning in the caudal extension is organized tonotopically and evidently involves the tectorium. In terms of the proportion of amphibian-papillar hair cells in the caudal extension, we report more diversity among 8 species of a single genus (Eleutherodactylus) on a single island (Puerto Rico) than has been found so far among all of the (more than 50) other modern anurans examined for this feature from around the world. These 8 Puerto Rican species have overlapping habitat and conspicuous diversity in the male advertisement call. For 7 of the 8 species, we report that the call has transient spectral components in the frequency range of the amphibian papilla, and that the proportion of caudal extension hair cells and the frequency distribution of those components are correlated. Thus one might conclude that the selective pressures that led to diversity of calls among the 8 species also led to diversity in form of the amphibian papilla.Abbreviations AP amphibian papilla - BEF best excitatory frequency - PR Puerto Rican - SEM scanning electron microscope - SVL snout-vent length - TC tectorial corner - TM tectorial membrane     

Organization of the superficial neuromast system in goldfish, Carassius auratus

Distribution, morphology, and orientation of superficial neuromasts and polarization of the hair cells within superficial neuromasts of the goldfish (Carassius auratus) were examined using fluorescence labeling and scanning electron microscopy. On each body side, goldfish have 1,800-2,000 superficial neuromasts distributed across the head, trunk and tail fin. Each superficial neuromast had about 14-32 hair cells that were arranged in the sensory epithelium with the axis of best sensitivity aligned perpendicular to the long axis of the neuromast. Hair cell polarization was rostro-caudal in most superficial neuromasts on trunk scales (with the exception of those on the lateral line scales), or on the tail fin. On lateral line scales, the most frequent hair cell polarization was dorso-ventral in 45% and rostro-caudal in 20% of the superficial neuromasts. On individual trunk scales, superficial neuromasts were organized in rows which in most scales showed similar orientations with angle deviations smaller than 45 degrees . In about 16% of all trunk scales, groups of superficial neuromasts in the dorsal and ventral half of the scale were oriented orthogonal to each other. On the head, most superficial neuromasts were arranged in rows or groups of similar orientation with angle deviations smaller than 45 degrees . Neighboring groups of superficial neuromasts could differ with respect to their orientation. The most frequent hair cell polarization was dorso-ventral in front of the eyes and on the ventral mandible and rostro-caudal below the eye and on the operculum.