Auditory nerve fibers in young and quiet-aged gerbils: morphometric correlations with endocochlear potential.

The number, size and distribution of myelinated nerve fibers were analyzed in the osseous spiral lamina (OSL) of young and old gerbils raised in a quiet environment. Because decreased endocochlear potentials (EPs) play a significant role in age-related hearing loss in the gerbil, we correlated morphometric and topographical data for nerve fibers with EP measurements in the same ear. Fibers were analyzed at the 2 and 10 kHz locations. The number of fibers at the 2 kHz location ranged from 12 to 47% greater than at the 10 kHz place in both young and aged specimens. No significant correlation was found between the number of fibers and the EP. Nerve fibers in gerbil tend to be distributed vertically by size within the OSL [Slepecky et al. (2000) Hear. Res. 144, 124-134], a result also found in cats and guinea pigs. Smaller fibers are more often found towards the scala vestibuli side of the OSL, whereas larger fibers are concentrated towards the scala tympani. The present data confirmed this distribution in young gerbils; however, in aged ears the distribution often became more uniform. Moreover, fiber distribution and ganglion cell size were highly correlated with EP. As EP declined, the fiber size distribution in the OSL became more uniform and the mean cross-sectional area of spiral ganglion cells and fiber diameter decreased. Thus, for whatever reason, certain indices of auditory nerve fiber morphometrics appear to be associated with the EP.

Anatomic and morphometric changes to gerbil posterior cristas following transtympanic administration of gentamicin and streptomycin.

Many studies have sought to document ototoxic damage and to study repair and regeneration of mammalian vestibular sensory epithelia. However, linear density analysis of the sensory cells or use of methods that focus on detection of actin in the stereocilia and cuticular plates at the reticular lamina detect only the disappearance of "hair cells" as defined by a narrow set of criteria. The research presented here focuses on the effects of two ototoxic drugs (gentamicin and streptomycin). We used light microscopic analysis of semithin sections to observe changes in the distribution of sensory and supporting cell nuclei and to elucidate other, previously undetected, morphological changes that occurred within the vestibular epithelia. Age-matched untreated and vehicle-treated controls showed that the gerbil posterior crista is asymmetrical on either side of the septum cruciatum; the longer end is taller and narrower than the shorter end. In cross sections taken throughout the length of each posterior crista, the thickness of the sensory epithelium along the sides (peripheral zone) is greater than at the apex (central zone). In tissue sections of the sensory epithelium, the ratio of sensory cell nuclei to support cell nuclei is slightly over 1:1.5 in all regions except the septum cruciatum where most sensory cells are absent and supporting cells predominate. In tissue sections from the most damaged drug-treated specimens, there was a decrease in the linear density of nuclei in the sensory cell layer, with a compensatory increase in the linear density of nuclei in the support cell layer of the sensory epithelia. In these specimens, linear density of total nuclei/tissue section remained the same. In these regions, the width of the epithelium became up to 50% thinner. The ratio of sensory to supporting cell nuclei changed to 1:6. Drug exposure led additionally to a decrease in length of the cristas, but there was not a linear relationship between the change in length of the crista and length of the septum cruciatum in these shorter cristas. In drug-treated cristas, other changes included a decrease in calculated surface area and volume of the epithelia. Thus, while linear density measurements of sensory cell nuclei provide an indication of damage, there are additional anatomic changes to the cristas and caution is advised with regard to interpreting changes as "loss" of cells.

Immunocytochemical identification of proteins within the Pacinian corpuscle.

Light- and electron-microscopic immunocytochemistry (ICC) was performed on Pacinian corpuscles (PCs) obtained from cat mesentery to determine the presence and location of various proteins within the accessory capsule and the neurite. Antibodies to tubulin, neurofilament 200, actin, collagen II and V, glial fibrillary acidic protein (GFAP) and S-100 were used. Type II collagen was localized only in the outer core of the accessory capsule, which is composed of an inner core, an intermediate layer or growth zone, an outer core and an external capsule. Type V collagen was found only in the intermediate growth zone. Intermediate filaments labeled with anti-GFAP were only found in the inner core. The calcium-binding protein that was labeled by anti-S-100 was found only in the inner core. Diffuse and variable staining for actin is present throughout the accessory capsule. The differences in distribution of these various proteins within the capsule suggest different structural/functional properties of the various capsule regions. The neurite was found to contain microtubules (i.e., tubulin) and neurofilaments throughout, but these cellular inclusions were not found within the cytoplasmic extensions (filopodia) that project from the neurite into the hemilamellar clefts formed by the inner-core hemilamellae. The extensions, however, were found to contain actin in a much greater density than that seen in the neurite proper. The presence of actin, but apparent lack of other cytostructural elements within the extensions, is highly reminiscent of the composition of stereocilia found on vestibular and auditory hair cells. Since stereocilia have been shown to play a role in hair-cell mechanotransduction, it is possible that the cytoplasmic extensions are significantly involved with mechanotransduction within the PC.

Study of afferent nerve terminals and fibers in the gerbil cochlea: distribution by size.

The purpose of the present study was to determine if the synaptic terminals and nerve fibers in the gerbil cochlea fall into morphologically and spatially classified groups. In cats and guinea pigs, these groups, based on size, location on inner hair cell (IHC) and stratification within the osseous spiral lamina, have been found to correlate with spontaneous rate, threshold sensitivity and projection pattern to the cochlear nucleus. Thus, there may be anatomical data to suggest mechanisms for intensity coding of different frequencies of sound. Afferent nerve terminals contacting IHCs in the gerbil cochlea were analyzed with regard to size and location. Data were obtained from serial thin sections (700 for each IHC) cut perpendicular to the long axis of eight IHCs (two apical and two basal IHCs from two cochleas), observed and photographed using a transmission electron microscope. Results indicate that the percentage of modiolar versus pillar-side terminals around each IHC varies from cell to cell. In some cases, the smallest fibers were located on the modiolar side, but a consistent distribution of the smallest fibers on this side of the cell was not characteristic. While a size-based segregation of terminals does not appear around the perimeter of the IHC, modest size-based segregation of nerve fibers is found in the osseous spiral lamina. Perimeter measurements were made from myelinated fibers cut in cross-section, obtained from semi-thin sections in the distal (near the IHCs) and proximal (near the spiral ganglion) regions of the osseous spiral lamina. Best-fit line analysis indicates there is a modest nerve fiber size/vertical organization along the scala tympani/scala vestibuli (SV) axis of the nerve bundles within the osseous spiral lamina such that more of the smaller perimeter fibers are located on the SV side and more of the larger perimeter fibers are located on the ST side. Our data for terminals at the IHC are different from those seen in the cat; our data for nerve fibers in the osseous spiral lamina support those seen in the cat and guinea pig.

Age-related changes in microtubules in the guinea pig organ of Corti. Tubulin isoform shifts with increasing age suggest changes in micromechanical properties of the sensory epithelium.

Biochemical and immunocytochemical analyses have been used to provide new insights into age-related changes in the sensory and supporting cells of the guinea pig organ of Corti. Quantitative densitometry of immunoblots showed that, while levels of alpha-tubulin remained relatively constant in guinea pigs from 3 weeks to 18 months old, there were progressive shifts in some tubulin isoforms. Levels of tyrosinated tubulin increased with age, nontyrosinatable tubulin (delta2-tubulin) showed a compensatory decrease, but detyrosinated tubulin did not change; acetylated, polyglutamylated, and glycylated tubulin levels also decreased. Immunolabeled tissue sections showed that cell type-specific distribution of tubulin seen in young guinea pigs (tyrosinated in the microtubules of the sensory cells, and post-translationally modified isoforms in the supporting cells) did not change as animals aged. However, there were age-related decreases in labeling for alpha-tubulin and all post-translationally modified isoforms. Biochemical and immunocytochemical results both support an age-related decrease in the number and/or length of microtubules as well as an increase in the pool of soluble tyrosinated and detyrosinated tubulin. They further suggest that microtubules containing nontyrosinatable tubulin from older animals are the sites for further modification of tubulin by acetylation, polyglutamylation, and glycylation. Changes in tubulin isoform levels and stability of microtubules in the organ of Corti may alter its micromechanical properties; the resulting changes in conduction of sound-induced vibration would provide one mechanism for age-related hearing loss.

Histologic changes after semicircular canal occlusion in guinea pigs.

HYPOTHESIS: Histologic changes occurring after varying degrees of surgical trauma to the inner ear in guinea pigs can reveal the mechanism of hearing preservation/loss. BACKGROUND: Surgical approaches to the inner ear that allow for hearing preservation have gained increasing acceptance in neurotologic surgery. The mechanisms responsible for hearing preservation and hearing loss after partial labyrinthectomy are as yet poorly understood. METHODS: Ten animals underwent semicircular canal occlusion, suctioning of perilymph, ampullectomy, or wide vestibulotomy. Tone-burst auditory brain stem response (ABR) thresholds were performed at weekly intervals after surgery. After 4 weeks, temporal bone specimens were processed to obtain 10-jim sections from plastic-embedded ears. The histologic findings were correlated with the initial and final ABR thresholds. RESULTS: After surgical occlusion of one or more semicircular canals, ABR thresholds were preserved, as the authors reported previously. Suctioning of inner ear fluid led to transient loss of thresholds with recovery. Ampullectomy produced dichotomous results, with some subjects preserving auditory function and others losing auditory function. Wide vestibulotomy resulted in permanent loss of auditory function in most cases. Histologically, there was intraluminal fibrosis and inflammation near the site of surgical entry. Most specimens showed normal cochlear architecture and hair cell counts, irrespective of the degree of hearing loss. Vestibular hair cells were also well preserved, even when they were close to the site of surgical injury. CONCLUSIONS: These findings suggest that electromechanical changes, rather than cell death, are responsible for changes in auditory and vestibular function after partial labyrinthectomy.

Comparison of vestibular and cochlear ototoxicity from transtympanic streptomycin administration.

HYPOTHESIS: The relative dose-related cochlear and vestibular ototoxicity produced by transtympanically injected streptomycin (SM) compared to that of gentamicin (GM) was assessed. BACKGROUND: Although SM, the first aminoglycoside used transtympanically, is thought to be selectively vestibulotoxic, it has been replaced by GM in current clinical use. Little experimental data exist that directly demonstrate the relative cochlear and vestibular ototoxicity resulting from transtympanic administration of SM compared to GM. METHODS: Histologic evaluation was performed on inner ears from Mongolian gerbils to study vestibular and cochlear damage. Comparisons were made between animals receiving single (1 x SM) and five daily (5 x SM) injections of SM/Gelfoam-slurry and similarly injected and noninjected controls. These data were compared to results obtained using GM (1 x GM and 5 x GM) reported previously. RESULTS: Two weeks after injection, parallel qualitative and quantitative changes were seen in posterior cristae and cochlear sensory epithelia in the 1 x and 5 x SM injected groups, similar to those resulting from GM injections. Statistically significant decreases in number of hair cells were seen when 5 x SM injected ears were compared to 1 x SM injected ears and control ears. Increased damage was seen with increased dosage of each drug. Whenever damage was observed to the posterior crista sensory cells, damage was also seen in cochlear hair cells. CONCLUSIONS: In this model, SM and GM produced significant cochlear damage when vestibular damage occurred. These results suggest that, in the gerbil, SM and GM are ototoxic but not selectively vestibulotoxic. Increasing the number of transtympanic injections generally increases the damage to sensory hair cells in the posterior crista and the cochlea. A variation in interanimal susceptibility to ototoxic effects exists, but the amount of damage is consistent in cochlear and vestibular hair cells from the same animal. No evidence for selective vestibular ototoxicity from transtympanic SM was found.

Olivocochlear neurons in the chinchilla: a retrograde fluorescent labelling study.

Although the chinchilla is widely used as a model for auditory research, little is known about the distribution and morphology of its olivocochlear neurons. Here, we report on the olivocochlear neurons projecting to one cochlea, as determined by single and double retrograde fluorescent tracer techniques. 10 adult chinchillas were anesthetized and given either unilateral or bilateral injections of a fluorescent tracer (either Fluoro-Gold or Fast Blue) into scala tympani or as a control, a unilateral injection into the middle ear cavity. The results indicate that there are similarities as well as significant differences between the chinchilla and other species of rodents in the distributions of their olivocochlear neurons. Based on three well-labelled cases, there was a mean total of 1168 olivocochlear neurons in the chinchilla. Of these, the majority (mean 787) were small, lateral olivocochlear neurons found almost exclusively within the ipsilateral lateral superior olivary nucleus. The next largest group consisted of a mean of 280 medial olivocochlear neurons virtually all of which were located in the dorsomedial peri-olivary nucleus. Chinchilla medial olivocochlear neurons were more predominantly crossed in their projections (4:1) than in any known species. The smallest group of olivocochlear neurons (mean 101) consisted of larger lateral olivocochlear neurons (shell neurons) which were located on the margins of the superior olivary nucleus and which projected mainly (2.2:1) ipsilaterally. Double retrograde labelling was observed only in medial olivocochlear neurons and occurred in only 1-2% of these cells. The results confirm previous findings which indicated a relative paucity of fibers belonging to the uncrossed as compared to the crossed olivocochlear bundle. This, together with the strong apical bias of the uncrossed projection reported previously, offers possible explanations for the apparent absence of efferent-mediated suppressive effects of contralateral acoustic stimulation in this species. Regarding the lateral olivocochlear system, the chinchilla is shown to possess both intrinsic and shell neurons, as in the rat.

Study of the gerbil utricular macula following treatment with gentamicin, by use of bromodeoxyuridine and calmodulin immunohistochemical labelling.

Effects of ototoxic drugs on the gerbil vestibular sensory epithelium were probed by use of immunocytochemical labelling with antibodies to both a mitogenic marker (bromodeoxyuridine) and a hair cell specific protein (calmodulin). Nine animals had gentamicin administered once daily for 5 days, as a transtympanic injection into the right middle ear. They additionally were given a daily intraperitoneal injection of bromodeoxyuridine, starting on the same day as the gentamicin injection and continuing until the day of sacrifice. Nine other animals, serving as controls for bromodeoxyuridine incorporation, received only the intraperitoneal injections of bromodeoxyuridine. The inner ears from three gerbils were obtained at 1, 2 or 4 weeks following the last gentamicin injection and utricles from the injected ears were processed for immunohistochemical analysis. In specimens where gentamicin was administered, we found evidence of bromodeoxyuridine incorporation in 17 cells (10 single cells and 7 pairs of cells) in a total of 216 sections taken from the central regions of the 9 utricles. However, in control specimens, no bromodeoxyuridine labelling was found in any cells of the 216 sections examined. Of 10 single cells labelled with bromodeoxyuridine, two cells in the hair cell layer were labelled with antibodies against calmodulin. One had a faint labelling in the nucleus and the other in the stereocilia, but not in the cell bodies. Of 7 pairs of cells, two pairs with nuclei localized in the hair cell layer had faint labelling for calmodulin in the nuclei, but no labelling in any other part of the cell. The other 13 cells labelled with antibodies to bromodeoxyuridine were not labelled with antibodies to calmodulin. Our results suggest that the bromodeoxyuridine-labelled cells could not be positively identified as hair cells based on immunohistochemical labelling for calmodulin.

Immunocytochemical localization of calmodulin in the vestibular end-organs of the gerbil.

This study demonstrates the presence of calmodulin in the vestibular end-organs of the gerbil by use of immunocytochemistry. Using fluorescence microscopy, calmodulin was localized to the cytoplasm, cuticular plate, and stereocilia of both type I and type II hair cells in the sensory epithelia of the utricle and cristae ampullaris; no label was found in the supporting cells, the dark cells, or the nerve fibers. There was no immunoreactive distinction between the labeling of type I and type II hair cells in the striolar or extrastriolar regions. Thus, immunocytochemical labeling for calmodulin provides a good marker for hair cells in gerbil vestibular epithelium. The presence of calmodulin in the stereocilia was confirmed by immunoelectron microscopy using secondary antibodies coupled to colloidal gold.

Localization of microtubules containing posttranslationally modified tubulin in cochlear epithelial cells during development.

In the adult gerbil inner ear, hair cell microtubules contain predominantly tyrosinated tubulin while supporting cell microtubules contain almost exclusively other isoforms. This cell-type specific segregation of tubulin isoforms is unusual, and in this respect the sensory and supporting cells in this sensory organ differ from other cells observed both in vivo and in vitro. Thus, we hypothesized there must be a shift in the presence and location of tubulin isoforms during development, directly associated with the onset of specialized functions of the cells. We describe the appearance and/or disappearance of tubulin isoforms in sensory hair cells and five different supporting cells (inner and outer pillar cells, Deiters cells, cells of Kölliker's organ, and cells of the tympanic covering layer) during development of the gerbil organ of Corti from birth to 14 days after birth. Tyrosinated tubulin was initially present in all cells and remained predominant in cells that decrease in number (Kölliker's organ and tympanic covering layer) and exhibit active processes such as secretion and motility (sensory cells). Posttranslational modifications occurred in the supporting cells in a time-dependent manner as the number and length of microtubules increased and development proceeded, but the establishment of elongated cell shape and polarity occurred prior to the appearance of acetylation, detyrosination, and polyglutamylation of tubulin. In the pillar and Deiters cells, posttranslational modifications progressed from cell apex to base in the same direction as microtubule elongation. In the pillar cells, posttranslational modifications occurred first at the apical surfaces. In the pillar cells, the appearance of acetylated tubulin was rapidly followed by the appearance of detyrosinated tubulin. In Deiters cells, the appearance of acetylated tubulin preceded the appearance of detyrosinated tubulin by one or more days. At onset of cochlear function, detyrosinated tubulin and acetylated tubulin had achieved their adult-like pattern, but polyglutamylated tubulin had not.

[Immunohistochemical labeling of inner ear tissues embedded in polyethylene glycol 4000--comparative study with araldite and unicryl embedded sections].

An improved method for embedding specimens in polyethylene glycol (PEG) 4000, a water soluble polymer, was used to prepare the vestibular end-organs of the inner ear. Staining of the tissue sections of PEG embedded specimens with antibodies to alpha-tubulin and to calmodulin was compared with staining of tissue sections of Araldite and Unicryl embedded specimens. PEG embedded sections revealed sensitive immunocytochemical labeling with excellent morphological resolution. The problems of embedding and orienting small specimens of the inner ear in PEG are described and the methods used to solve them are described.

Cytoskeletal and calcium-binding proteins in the mammalian organ of Corti: cell type-specific proteins displaying longitudinal and radial gradients.

Whole mounts and tissue sections of the organ of Corti from two representative mammalian species, the Mongolian gerbil (Meriones unguiculatus) and the guinea pig (Cavea porcellus) were probed with antibodies to cytoskeletal and calcium-binding proteins (actin, tubulin, including post-translational modifications, spectrin, fimbrin, calmodulin, parvalbumin, calbindin, S-100 and calretinin). All of the proteins tested were expressed in both species. New findings include the following. Actin is present in large accumulations in cell bodies of the Deiters cells under the outer hair cells (OHC), as well as in the filament networks previously described. These accumulations are more prominent in the apical turns. Tubulin is present in sensory cells in the tyrosinated (more dynamic) form, while tubulin in the supporting cells is post-translationally modified, indicating greater stability. Fimbrin, present in the stereocilia of both IHCs and OHCs, is similar to the isoform of fimbrin found in the epithelial cells of the intestine (fimbrin-I), which implies that actin bundling by fimbrin is reduced in the presence of increased calcium. Parvalbumin appears to be an IHC-specific calcium-binding protein in the gerbil as well as in the guinea pig; labeling displays a longitudinal gradient, with hair cells at the apex staining intensely and hair cells at the base staining weakly. Calbindin displays a similar longitudinal gradient, with staining intense in the IHCs and OHCs at the apex and weak to absent in the base. In the middle turns of the guinea pig cochlea, OHCs in the first row near the pillar cells lose immunoreactivity to calbindin before those in the second and third rows. Calmodulin is found throughout the whole cochlea in the IHCs and OHCs in the stereocilia, cuticular plate, and cell body. Calretinin is present in IHCs and Deiters cells in both species, as well as the tectal cell (modified Hensen cell) in the gerbil. S-100 is a supporting cell-specific calcium-binding protein which has not been localized in the sensory cells of these two species. The supporting cells containing S-100 include the inner border, inner phalangeal, pillar, Deiters, tectal (in gerbil) and Hensen cells, where labeling displays a longitudinal gradient decreasing in intensity towards the apex (opposite to what has been seen with labeling for other proteins in the cochlea).

Post-translational modifications of tubulin suggest that dynamic microtubules are present in sensory cells and stable microtubules are present in supporting cells of the mammalian cochlea.

Post-translational modifications to tubulin in the sensory and supporting cells of the cochlea were studied using antibodies specific to the tyrosinated, detyrosinated, acetylated and polyglutamylated isoforms. In the sensory cells, microtubules which label intensely with antibodies to tyrosinated tubulin are found in networks within the cytoplasm. Microtubules which label with antibodies to detyrosinated tubulin and polyglutamylated tubulin, but not acetylated tubulin, form a small component of the microtubules found in the cytoplasm only in the region below the cuticular plate. Microtubules in the supporting cells (inner and outer pillar cells and Deiters cells) are arranged in bundles and contain little tyrosinated tubulin. They are composed instead of predominantly post-translationally modified isoforms which include detyrosinated, acetylated and polyglutamylated tubulin. The findings suggest that microtubules in the sensory cells form dynamic structures, since microtubules that undergo cyclic polymerization and depolymerization predominantly contain tubulin that has not yet had its carboxy-terminal tyrosine residue removed. The presence of microtubules in the supporting cells in which the tubulin has been polymerized into microtubules long enough to be post-translationally modified, provides evidence that these microtubules are stable, long-lived and could contribute to the structural support of the sensory organ of Corti.

A simplified method for obtaining 0.5-microns sections of small tissue specimens embedded in PEG.

We describe a new method for embedding small specimens in polyethylene glycol (PEG) 4000. This method preserves cell morphology and provides sensitive immunocytochemical labeling with excellent subcellular resolution. Small tissues are embedded in agarose so that they can be grouped together and oriented for sectioning before infiltration with PEG 4000, a water-soluble polymer. Fixation, embedding, sectioning, and staining can be performed in 1 day. Results from immunocytochemical studies localizing actin and tubulin on 0.5-micron sections of PEG-embedded specimens are compared with those obtained on semi-thin sections of araldite-embedded specimens and demonstrate the ease, speed, and increased sensitivity of this embedding method.

Immunocytochemical comparison of posttranslationally modified forms of tubulin in the vestibular end-organs of the gerbil: tyrosinated, acetylated and polyglutamylated tubulin.

Specific antibodies against alpha-tubulin, acetylated alpha-tubulin, tyrosinated alpha-tubulin and polyglutamylated alpha- and beta-tubulin were used to compare the distribution of posttranslationally modified tubulin in the vestibular end-organs of the gerbil. Antibodies to acetylated tubulin labeled a dense network of microtubules in the hair cells and bundles of microtubule in the supporting cells. Nerve fibers within and below the epithelium were weakly labeled. This localization paralleled that seen with antibodies to alpha-tubulin which labeled all microtubules present in the cells. Antibodies to tyrosinated tubulin labeled networks and bundles of microtubules in both hair cells and supporting cells and in addition gave intense, diffuse labeling in the cytoplasm of both cell types. It also labeled the nerve fibers. Antibodies to polyglutamylated tubulin were localized mainly in nerve fibers, and in the calyces the labeled microtubules were found running circumferentially around the type I sensory hair cells. Thus, tyrosinated tubulin was found in the fine networks of microtubules in both the sensory and supporting cells. Acetylated tubulin was found in the dense networks and bundles of microtubules in the sensory and supporting cells, but did not colocalize with polyglutamylated tubulin, which was found predominantly in the nerve fibers. The labeling patterns for the tyrosinated tubulin and posttranslationally modified tubulins in the sensory and supporting cells of the vestibular end organs differ from that seen in the organ of Corti and may reflect differences in the stability of the microtubules and the mechanical properties of the sensory epithelium.

Three microtubule-organizing centres collaborate in a mouse cochlear epithelial cell during supracellularly coordinated control of microtubule positioning.

Large cell surface-associated microtubule bundles that include about 3,000 microtubules assemble in certain epithelial cells called inner pillar cells in the mouse organ of Corti. Microtubule-organizing centres (MTOCs) at both ends and near the middle of each cell act in concert during control of microtubule positioning. In addition, the three cell surface-associated microtubule-organizing centres are involved in coordinating the connection of bundle microtubules to cytoskeletal components in neighbouring cells and to a basement membrane. The precisely defined locations of the three MTOCs specify the cell surface regions where microtubule ends will finally be anchored. The MTOCs are modified as anchorage proceeds. Substantial fibrous meshworks assemble at the surface sites occupied by the MTOCs and link microtubule ends to cell junctions. This procedure also connects the microtubule bundle to cytoskeletal arrays in neighbouring cells at two of the MTOC sites, and to the basilar membrane (a substantial basement membrane) in the case of the third site. A fourth meshwork that is not positioned at a major MTOC site is involved in connecting one side of the microtubule bundle to the cytoskeletons of two other cell neighbours. The term surfoskelosome is suggested for such concentrations of specialized cytoskeletal materials and junctions at cell surface anchorages for cytoskeletal arrays. The large microtubule bundle in each cell is composed of two closely aligned microtubule arrays. Bundle assembly begins with nucleation of microtubules by a centrosomal MTOC that is attached to the apical cell surface. These microtubules elongate downwards and the plus ends of many of them are apparently captured by a basal MTOC that is attached to the plasma membrane at the bottom of the cell. In the lower portion of the cell, the microtubule bundle also includes a basal array of microtubules but these elongate in the opposite direction. This investigation provides evidence that they extend upwards from the basal MTOC to be captured by a medial MTOC which is attached to the plasma membrane and situated near the mid-level of the cell. However, there are substantial indications that the basal array's microtubules are also nucleated by the apically situated centrosomal MTOC, but escape from it, and are translocated downwards for capture of their plus ends by the basal MTOC. If this is the case, then these microtubules continue to elongate after translocation and extend back up to the medial MTOC, which captures their minus ends.

Expression of actin isoforms in the guinea pig organ of Corti: muscle isoforms are not detected.

The specificity of antibodies to actin was assayed by use of immunoblots and histological sections of control tissues enriched for each of six different isoforms. On immunoblots, all antibodies stained at most one band of protein in most of the control materials, with a molecular weight of approximately 43 kDa. Their pattern of staining of muscle and nonmuscle tissues indicated their isoform specificity. On tissue sections, immunocytochemical staining demonstrated cellular and subcellular localization of the different isoforms. Once characterized with regard to specificity, these antibodies were used to probe actin in the guinea pig organ of Corti. None of the four muscle isoforms of actin were found in either immunoblots or tissue sections of the organ of Corti. Both beta- and gamma-cytoplasmic isoforms of actin were present in hair cells and supporting cells. This leaves open to investigation the role which cytoplasmic actins play in these cells of the organ of Corti.

Semi-serial electron-micrographic reconstruction of putative transducer sites in Pacinian corpuscles.

The Pacinian corpuscle (PC) is composed of an afferent neurite surrounded by an accessory capsule formed by concentric layers of lamellae. Projecting from the neurite, which is elliptical in cross-section, are "filopodia" or axonal, spike-like extensions. These filopodia are the putative sites of transduction. It has been proposed that two populations of filopodia organized in morphofunctional opposition exist, and that this arrangement is responsible for the bidirectional sensitivity of PCs as seen in receptor potential recordings. In order to explore this possibility, PCs obtained from cat mesentery were processed for electron microscopy, and semiserial reconstructions were made. We evaluated the extensions' (n > 110) locations, inclusions, shapes, and sizes. The filopodia were found to project along the major elliptical axis of the neurite, their density being approximately 2.8 per micron. The filopodia were found to contain filaments, vesicles, and amorphous ground substance, and dense accumulations of mitochondria were found at their bases. Measurements of their size (i.e., length, width, and height) suggest that there are two different types of filopodia. No other obvious relations among filopodial type, location along the neurite, and landmarks for transduction were found. The presence of the two filopodial types may be the basis for the bidirectional sensitivity of the PC.