Surface Of Hair Cells Research Articles

A newly identified surface coat on cochlear hair cells.

Routine electron microscope methods do not well preserve or stain the surface coat or glycocalyx on cochlear hair cells. In other tissues, enhanced preservation and staining of these glycoconjugates was obtained following fixation with glutaraldehyde containing a cationic dye (e.g., Alcian blue and ruthenium red). When cochleas were fixed with glutaraldehyde containing Alcian blue, the endolymphatic surface of hair cells, but not the supporting cells, displayed an extensive (approximately 90 nm thick) surface coat. Alcian blue positive material was also observed in the tectorial and basilar membranes and in a portion of the spiral ligament. In addition, acellular bands of Alcian blue positive material were observed between the tectorial membrane and the reticular lamina or inner sulcus cells. Although the function of these cochlear glycoconjugates is not yet known, it is proposed that they serve to attach the tectorial membrane to the organ of Corti, and they are involved in stereocilia fusion following sound exposure and ototoxic drug administration.

Freeze-fracture study of the vestibular hair cell surface during development.

The ciliary arrangement and external surface of the vestibular receptor cell were studied in their immature stages. In the first stages, immature stereocilia are similar to the microvilli of the adjacent supporting cells. Later, when the cilia grow, a geometric arrangement of the stereocilia occurs, while the peripheral microvilli disappear. The size of the hair cell apex increases. The biggest and more bulbous cells correspond to type I hair cells, but in the newborn cat it is difficult to distinguish accurately type II flat hair cells from immature hair cells.

Ionic environment of cochlear hair cells.

The scala media of the adult cochlea in mammals comprises a morphologically closed compartment sealed with tight junctions of the intermediate to tight types. The unique ionic composition of endolymph is maintained by the stria vascularis through active reabsorption of sodium and active secretion of potassium against ionic gradients. The subtectorial space is only a partially closed compartment which communicates with the endolymph via holes in the tectorial membrane at its outer insertion to the organ of Corti. Hardesty's membrane divides the subtectorial space into two compartments: one facing the surfaces of inner hair cells and one facing the surfaces of outer hair cells. In the study of comparative anatomy, hair cells, e.g. in the lizard, basilar papilla are of two types: those covered with a tectorial membrane and those being free-standing lacking the tectorial membrane. The ionic environment of the hair cell surface seems to be the same, independent of whether covered with a tectorial membrane or not. The tectorial membrane itself is semipermeable to ions in the endolymphatic space. Only the surface structures of the hair cell with the sensory hairs facing the subtectorial space are exposed to the high concentration of potassium, whereas the remaining parts of the hair cell are surrounded by a fluid having a more normal extracellular type of ionic composition (cortilymph/perilymph). During embryonic development the ionic composition of endolymph develops in parallel with the morphologic maturation of the stria vascularis. A completely mature composition of endolymph is reached before any electrophysiological potentials in the cochlea can be elicited. The sensory hair surface of hair cells has reached a mature morphology prior to the maturation of endolymph. In several species the tectorial membrane is morphologically only partially mature when the increase of the potassium concentration of endolymph starts. Drugs primarily affecting the stria vascularis causing a transient change of the ionic composition of endolymph result in a transient dysfunction of inner ear potentials. If the ionic changes persist for longer time, morphological changes can occur in both the stria vascularis and the hair cells of the organ of Corti. Whether such changes are primarily caused by the ototoxic drug itself or by changes in the ionic composition of endolymph has to be explored further.

Development of the auditory hair cell surface in human fetuses. A scanning electron microscopy study.

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.

Cytoskeletal architecture of the chicken hair cells revealed with the quick-freeze, deep-etch technique.

Replicas of the apical surface of hair cells of the inner ear (vestibular organ) were examined after quick-freeze, deep-etch and rotary shadowing. With this technique we demonstrate how actin filaments are organized and associated with the plasma membrane in the stereocilia and cuticular plate as well as inside the junctional complex. In each stereocilium there are thread-like connectors running from the actin filament bundle to the limiting membrane. Many of the actin filaments in the cuticular plate are connected to the apical cell membrane by tiny branched connecting units like a 'crow's foot'. Where these 'feet' contact the membrane there is a small swelling. These branched 'feet' extend mainly from the ends of the actin filaments but some connect the lateral surfaces of the actin filaments as well. Actin filaments in the cuticular plate are also connected to each other by finer filaments, 3 nm in thickness and 74 +/- 14 nm in length. These 3 nm filaments (which measure 4 nm in replicas) connect actin filaments, not only of the same polarity, but of opposite polarities as documented by examining replicas of the cuticular plate which had been decorated with subfragment 1 (S1) of myosin. At the apicolateral margins of the cell we find two populations of actin filaments, one just beneath the tight junction as a network, the other at the level of the intermediate junction as a ring. The latter is composed of actin filaments that run parallel to each other; adjacent filaments often show opposite polarities, as evidenced by S1 decoration. Because of the polarity of the filaments this ring may be a 'contractile' ring.

A Study of the Nerve Endings of the Outer Hair Cells in the Organ of Corti: Using Scanning Electron Microscopy

The nerve endings on the outer hair cells of the guinea pig organ of Corti were studied using a scanning electron microscope. The nerve endings in the basal turn were entirely exposed in the space of Nuel, whereas in the upper turns almost all nerve endings were covered by the Deiters' cell bulge. The number and the size of the nerve endings gradually decreased from the basal turn toward the apical turn. The shape of the nerve endings was spindle-shaped in the basal turn, while it was round in the upper turn. Some nerve endings were found to locate close to the outer hair cell surface. No pathological change was observed in the nerve endings 25 hr after completion of series treatments of streptomycin or kanamycin. However, the outer hair cells consistently showed shrinkage, especially in the basal turn.

Cytodifferentiation of cochlear hair cells

Cytodifferentiation of a limited number of cochlear hair cells in the mouse starts on the 14th gestational day. One day later, the number of identified hair cells increases considerably. Cytodifferentiation apparently occurs in a gradient from the hair cell surface to the base. First, the irregular microvilli covering the future hair cell surfaces begin to show a regular pattern but are of the same thickness and length as microvilli on supporting cells. Second, a polarization of sensory hairs occurs with a stepwise increase in stereocilia length in the different rows toward the kinocilium. Finally, the cuticle is formed, giving an anchorage for sensory hair rootlets in the hair cell. At birth some hair cells can be found with immature surface morphologic features, e.g., stereocilia of the same length on the entire hair cell surface and lack of a cuticular plate. The onset of hair cell differentiation takes place without morphologic contact with ingrowing nerve fibers.

Principles in embryonic development and differentiation of vestibular hair cells.

The early development and maturation of vestibular hair cells in the CBA/CBA mouse were analyzed at the ultrastructural level with conventional transmission electron microscopy and freeze fracturing. Cells differentiating into future hair cells pass their terminal mitosis close to the otocyst lumen. The earliest morphologic sign of differentiation into future hair cells was the arrangement of microvilli in a regular fashion. Hair cell cytodifferentiation occurred with a gradient from the hair cell surface to the base. In parallel with the maturation of sensory hairs a structural intracellular rebuilding occurred: the number of polyribosomes and amount of rough endoplasmic reticulum decreased, the nucleus moved basally, and mitochondria accumulated in the supranuclear region. Both tight junctions and gap junctions occurred initially on developing hair cells. From the sixteenth gestational day on, gap junctions disappeared, indicating an uncoupling phenomenon. Afferent nerve terminals developed before efferent nerve endings. Maturation of innervation occurred, for the most part, postnatally, and after that hair cells reached mature morphology.

Scanning electron microscopic study of impulse noise—Induced mechanical damage in the cochlea

Binaural chinchillas were exposed at normal incidence to 160 dB peak SPL impulse noise at the rate of 2 impulses per min for 50 min. Animals were sacrificed at post‐exposure times varying from t = 0 through 30 days. The cochleas were prepared for SEM observation using a standard protocol. Immediately following exposure, a large (6‐mm) area of the organ of Corti was separated from its attachments to the basilar membrane along a fracture line that follows the outer pillar cells. The separated portion of the organ of Corti is left floating in the scala media with both ends attached to viable portions of the remaining sensory epithelia. Surprisingly, in the denuded areas of the basilar membrane, the inner hair cell surface structure remains comparatively normal during the early post‐exposure times while outer hair cells in the region bordering the main lesion show considerable changes in cilia structure and in the appearance of the reticular lamina‐cuticular plate complex. Scar formation and the absorption/phagocytosis of the free‐floating portions of the organ of Corti will be described, as well as the differing susceptibilities of inner and outer hair cell cilia to morphological changes. [Work supported by U.S. Army Med R and D Command, DAMD 17‐80‐C‐0133.]

Postnatal maturation of cochlear sensory hairs in the mouse.

The surface morphology of inner hair cells in the cochlea of the mouse is fully developed at birth. At this time a regular pattern of elongated microvilli covers the surfaces of outer hair cells at all levels of the cochlea. The sensory hair on outer hair cells undergo a morphological maturation during the first days postnatally. A mature surface morphology is reached 5-7 days after birth. There are two gradients in hair cell maturation in the cochlea: (1) basal to apical, and (2) from inner hair cells to the 3rd row of outer hair cells. Polarization of sensory hairs on an outer hair cell, i.e. polar differentiation of the sensory hairs in the radial direction, occurs as a stepwise increase in length of those facing the stria vascularis.

Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear.

Replicas of the apical surface of hair cells of the inner ear (vestibular organ) were examined after quick freezing and rotary shadowing. With this technique we illustrate two previously undescribed ways in which the actin filaments in the stereocilia and in the cuticular plate are attached to the plasma membrane. First, in each stereocilium there are threadlike connectors running from the actin filament bundle to the limiting membrane. Second, many of the actin filaments in the cuticular plate are connected to the apical cell membrane by tiny branched connecting units like a "crow's foot." Where these "feet" contact the membrane there is a small swelling. These branched "feet" extend mainly from the ends of the actin filaments but some connect the lateral surfaces of the actin filaments as well. Actin filaments in the cuticular plate are also connected to each other by finer filaments, 3 nm in thickness and 74 +/- 14 nm in length. Interestingly, these 3-nm filaments (which measure 4 nm in replicas) connect actin filaments not only of the same polarity but of opposite polarities as documented by examining replicas of the cuticular plate which had been decorated with subfragment 1 (S1) of myosin. At the apicolateral margins of the cell we find two populations of actin filaments, one just beneath the tight junction as a network, the other at the level of the zonula adherens as a ring. The latter which is quite substantial is composed of actin filaments that run parallel to each other; adjacent filaments often show opposite polarities, as evidenced by S1 decoration. The filaments making up this ring are connected together by the 3-nm connectors. Because of the polarity of the filaments this ring may be a "contractile" ring; the implications of this is discussed.

Development of otoconia

The development of the majority of otoconia in the macula utriculi occurs on the fifteenth and sixteenth gestational days (CBA/CBA strain of mice). The epithelial cell layer shows extensive protrusion of some cells, cytoplasmic blebbing from the surface of both hair cells and supporting cells, and electron dense inclusion bodies and vesicles surrounded by a double membrane in the apical part of supporting cells. This may reflect the formation of otoconia of varying sizes. Otoconia have an organic matrix that often shows a fibrillar substructure early in development. The majority of the organic aggregations that probably become the future otoconia are surrounded by a limiting membrane. The hexagonal shape of the otoconia can be observed as early as the sixteenth to seventeenth gestational days. Some otoconia show a nucleus-like focus in the center of the organic substance. Elemental mapping of calcium on the sixteenth gestational day shows an accumulation of calcium not only in the otoconia but also in the apical part of the epithelia cell layer below the otoconia. The endolymphatic space does not contain much calcium. These findings indicate that during the incorporation of calcium into otoconia, calcium is likely to emanate largely from the surface cells of the utricle.

Ionic basis of the receptor potential in a vertebrate hair cell.

Vertebrate hair cells, the primary receptors of auditory, vestibular and lateral-line organs, occur in epithelia which separate fluids of differing ionic composition. The apical surfaces of hair cells, on which the mechanosensitive hair bundles are situated, face a high-K+ fluid (termed endolymph in the inner ear); the basolateral surfaces instead contact fluid (perilymph or a related substance) of a composition similar to that of other extracellular fluids1–3. The universal occurrence of high-K+ fluid on the apical surfaces of hair cells in vertebrates has been taken as evidence that it is important for the transduction process, in particular that it relates to the ionic specificity4 of the conductance change5 underlying the receptor potential. There is, however, conflicting experimental evidence regarding this specificity. K+ has generally been thought to carry the receptor current, as replacement of endolymph with perilymph in the guinea pig cochlea abolishes the extracellularly recorded microphonic potential6. Yet microphonic potentials, as well as intracellular receptor potentials, have been recorded in other preparations when the apical surfaces of the hair cells faced instead a high-Na+ saline, and thus when the electrochemical gradient for K+ was near zero5,7. Ca2+ has also been proposed to carry the receptor current8, but its concentration is quite low in endolymph3, particularly that of the mammalian cochlea9. We present evidence here that the receptor current in a vertebrate hair cell is carried in vivo by K+, but that the transduction channel is in fact nonspecific, being permeable to Li+, Na+, K+, Rb+, Cs+, Ca2+, and at least one small organic cation.

Vestibular end organ mapping in the squirrel monkey.

Vestibular end organ mapping (surface preparation) was performed in squirrel monkeys by using succinic dehydrogenase staining. Comparing sensory hair cell counts and surface area measurements of the squirrel monkey to those of man and the guinea pig, the values obtained from the squirrel monkey were between those of man and the guinea pig. When regional (central versus peripheral) cell density was compared, the density of the peripheral area was 1.62X more on the average than that of the central portion.

The tectorial membrane of the rat.

AbstractHistochemical, x‐ray analytical and scanning and transmission electron microscopical procedures have been utilized to determine the chemical nature, physical appearance and attachments of the tectorial membrane in normal rats and to correlate these results with biochemical data on protein‐carbohydrate complexes. Additionally, pertinent histochemical and ultrastructural findings in chemically sympathectomized rats are considered. The results indicate that the tectorial membrane is a viscous, complex, colloid of glycoprotein(s) possessing some oriented molecules and an ionic composition different from either endolymph or perilymph. It is attached to the reticular laminar surface of the organ of Corti and to the tips of the outer hair cells; it is attached to and enclose the hairs of the inner hair cells. A fluid compartment may exist within the limbs of the “W” formed by the hairs on each outer hair cell surface. Present biochemical concepts of viscous glycoproteins suggest that they are polyelectrolytes interacting physically to form complex networks. They possess characteristics making them important in fluid and ion transport. Furthermore, the macromolecular configuration assumed by such polyelectrolytes is unstable and subject to change from stress or shifts in pH or ions. Thus, the attachments of the tectorial membrane to the hair cells may play an important role in the transduction process at the molecular level.

Scanning electron microscopy of the organ of corti after intense auditory stimulation: Effects on stereocilia and cuticular surface of hair cells

The organ of Corti of cats, guinea pigs and chinchillas exposed to pure tones of high intensity were first mounted on a slide in glycerol and mapped with light/phase contrast microscopy. Subsequently the same specimens were rinsed in water, freeze-dried and prepared for scanning electron microscopy. The study shows that it is well possible to use the same preparation for light microscopic and scanning electron microscopic investigations. Scanning electron microscopy permits detailed studies of the surface topography of the organ of Corti and demonstrates structural changes not visible in light/phase contrast microscopy. The changes are found on both inner and outer hair cells and include disarrangement of hairs, fusion of stereocilia and the formation of giant hairs, which exceed the normal stereocilia in length and thickness. The giant hairs are seen most frequently in cats following low frequency exposure. Moreover, the cuticular plates of the sensory cells are often extensively deformed. It is doubtful if these cells, which may look normal as studied by light/phase contrast microscopy, are still functional. In areas showing epithelial damage an increase in the number and size of the microvilli of the Deiters cells is often found.

ACETYLCHOLINESTERASE IN THE ORGAN OF CORTI

Histochemical investigation of acetylcholinesterase in the organ of Corti has been carried out by means of the Karnovsky's method. Acetylcholinesterase is located on the bottom of outer hair cells, tunnel radial fibers (upper), tunnel spiral bundle and inner spiral bundle. No reacrion was found on the bottom of inner hair cells, lower tunnel radial fibers, outer spiral bundle and bottom of outer hair cells on the outer raw at upper turns. In the bottom of outer hair cells, only vesiculated nerve endings showed the activity of acetylcholinesterase on their plasma membrane. In the basal and middle turns, it is assumed that the reaction products or enzyme from vesiculated nerve endings diffused to non-vesiculated nerve endings.On the endolymphatic surface of hair cells, acetylcholinesterase activity was also found but was lower than the activity on the vesiculated nerve endings at the bottom of the hair cells. In addition, some reductive reaction was found on the surface of the hair cells.

Activity of acetylcholinesterase on the endolymphatic surface of outer hair cells.

By means of the Karnovsky's modification of Koelle's thiocholine method for acetylcholinesterase (AChE), it has been demonstrated electronmicro-scopically that AChE is located on the endolymphatic surface of outer hair cells and not on the supporting cells in the organ of Corti of the guinea pig cochlea. The tissue from the middle part of the basal turn was used in all experiments. The identification of AChE was confirmed by use of appropriate concentration of selective inhibitors of cholines-terase. From the inhibitory effect of fixatives on AChE activity, it was obvious that the activity on the surface of outer hair cells was lower than the activity on the large nerve endings at the bottom of the hair cells.

The cnidoblast-musculoepithelial cell complex in the tentacles of hydra

The cnidoblast of hydra is known to be both a receptor and an effector cell. This paper describes a specialized cell complex in the tentacles of hydra in which the cnidoblast is shown to have a peculiar relationship to the musculoepithelial cell of the ectoderm. The musculoepithelial cell, which is shown for the first time to have myofilaments of two dimensions, also has a highly specialized area of attenuated cytoplasm, the plasmalemma of which makes a peculiar contact with numerous cnidoblasts on one side and with the acellular mesogloea on the other. It is proposed that the musculoepithelial cell serves an integrative function for the activation of its associated cnidoblasts and perhaps for those of other batteries. Intimately associated with this cell complex are numerous neuronal processes and neurosecretory fibers. The receptor element of the cnidoblast, the cnidocil, is described in detail and shown to bear a striking resemblance to the specialized apical surfaces of hair cells in the lateral line organ, organ of Corti and semicircular canals of vertebrates. Speculations as to the mode of action of the various elements of this complex are offered.