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.

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.

Evidence for calcium-binding proteins and calcium-dependent regulatory proteins in sensory cells of the organ of Corti.

Calcium is thought to play a major signaling role in outer hair cells to control metabolism, cytoskeletal integrity, cell shape and cell excitability. For this to happen, in resting cells the concentration of free calcium ions must be maintained at low levels so that focal increases can trigger specific events. In this paper, the localization of calcium, calcium-binding and calcium-dependent regulatory proteins in sensory cells from the guinea pig inner ear was demonstrated using immunocytochemical and histochemical techniques. We found the calcium buffer and/or calcium sensor proteins calmodulin, calbindin and calsequestrin predominantly in sensory cells and that when present, these proteins can be enriched in the outer hair cells. Calmodulin is found in the stereocilia, in the cuticular plate and in the cytoplasm and calbindin is found only in the cuticular plate and cytoplasm of both the inner and outer hair cells. The staining for these proteins in the outer hair cells is homogeneous, with no apparent compartmentalization along the lateral wall. Calsequestrin, thought to store and release calcium from membrane bound intracellular storage sites is found only in the cytoplasm of outer hair cells. There, it has a more punctuate staining pattern than does calmodulin or calbindin suggesting that it may be present in calciosomes rather than soluble in the cytoplasm. We did not detect caldesmon and S-100. Using the potassium pyroantimonate technique, we found precipitates containing calcium ions distributed throughout the cytoplasm of outer hair cells, with no evidence that the subsurface cisterns along the lateral wall act as calcium storage sites. Thus, calcium in resting cells is found in the cytoplasm along with calbindin and calmodulin and appears to have a punctate distribution consistent with a co-localization with calsequestrin. The implications of this distribution with respect to the slow shortening and elongation seen in outer hair cells are discussed.

Actin-binding and microtubule-associated proteins in the organ of Corti.

Actin-binding and microtubule-associated proteins regulate microfilament and microtubule number, length, organization and location in cells. In freeze-dried preparations of the guinea pig cochlea, both actin and tubulin are found in the sensory and supporting cells of the organ of Corti. Fodrin (brain spectrin) co-localized with actin in the cuticular plates of both inner and outer hair cells and along the lateral wall of the outer hair cells. Alpha-actinin co-localized with actin in the cuticular plates of the hair cells and in the head and foot plates of the supporting cells. It was also found in the junctional regions between hair cells and supporting cells. Profilin co-localized with actin in the cuticular plates of the sensory hair cells. Myosin was detected only in the cuticular plates of the outer hair cells and in the supporting cells in the region facing endolymph. Gelsolin was found in the region of the nerve fibers. Tubulin is found in microtubules in all cells of the organ of Corti. In supporting cells, microtubules are bundled together with actin microfilaments and tropomyosin, as well as being present as individual microtubules arranged in networks. An intensely stained network of microtubules is found in both outer and inner sensory hair cells. The microtubules in the outer hair cells appear to course throughout the entire length of the cells, and based on their staining with antibodies to the tyrosinated form of tubulin they appear to be more dynamic structures than the microtubules in the supporting cells. The microtubule-associated protein MAP-2 is present only in outer hair cells within the organ of Corti and co-localizes with tubulin in these cells. No other MAPs (1,3,4,5) are present. Tau is found in the nerve fibers below both inner and outer hair cells and in the osseous spiral lamina. It is clear that the actin-binding and microtubule-associated proteins present in the cochlea co-localize with actin and tubulin and that they modulate microfilament and microtubule structure and function in a manner similar to that seen in other cell types. The location of some of these proteins in outer hair cells suggests a role for microfilaments and microtubules in outer hair cell motility.

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.

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).

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.

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.

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.

Tectorial membrane. I: Static mechanical properties in vivo.

Although the tectorial membrane in the mammalian cochlea plays a crucial role in hair-cell stimulation, its mechanical properties have never been investigated under conditions approximating those under which it normally functions. For this reason, we performed such investigations in live Mongolian gerbils. Access to the tectorial membrane was gained through the lateral wall in the second cochlear turn. As far as possible, sodium ions were kept away from the tectorial membrane by avoiding injury to Reissner's membrane, relieving the perilymphatic pressure, and rinsing the scala media with an isotonic KCl solution. The tectorial membrane was manipulated with a flexible micropipette in three, approximately orthogonal, directions. Under these conditions the membrane was found to be highly compliant and resilient, and to have a relatively high tensile strength. Its viscosity was low. Some of these attributes were altered by sodium ions, dyes, or death.

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.

Ionic coupling among cells in the organ of Corti.

Gap junctions have been demonstrated morphologically among the supporting cells of the mammalian organ of Corti but, in contradistinction to reptiles, evidence for their existence between the supporting cells and hair cells is equivocal. The literature is ambiguous with respect to electrical coupling and dye coupling among the supporting cells, and no coupling of either kind has been demonstrated for the hair cells. We found strong coupling of both kinds among the supporting cells in the cochleas of live Mongolian gerbils and a less stable coupling between the supporting cells and the outer hair cells. The electrical coupling was established by recording alternating receptor potentials in the hair cells and following their decrement in the population of Hensen's cells; the dye coupling, by injecting Lucifer yellow electrophoretically into the hair cells or the supporting cells and investigating its spread to the neighboring cells. The electrical recordings were made by means of microelectrodes filled with either 1.5 or 3 M KCl or 1 M LiCl with 6% Lucifer yellow, the latter used for dye injection. The electrode resistances ranged from about 20 to 60 M omega in the first instance, and from about 50 to 110 M omega, in the second. The electrodes were inserted into the organ of Corti through scala media according to the method of Dallos, Santos-Sacchi and Flock (1982) modified by us. The alternating potential in Hensen's cells was usually larger than in the outer tunnel of Corti and remained practically constant up to the outer margin of the Hensen's-cell population. Its phase was the same as in the outer hair cells. When the dye was injected into a Hensen's cell, it always spread to neighboring Hensen's cells and often to Deiter's cells. Dye injected into outer hair cells (identified according to anatomical and physiological criteria) also spread to Deiter's and Hensen's cells and, usually, to other outer hair cells. Stained cells were identified in surface preparations and, on two occasions, in serial sections from plastic embedded cochleas.

Development of mature microcystic lesions in the cochlear nuclei of the Mongolian gerbil, Meriones unguiculatus.

Microcystic lesions are a persistent final stage in a neurodegenerative disorder characteristic of the cochlear nuclei of gerbils. When gerbils of various ages raised under known acoustic conditions were examined, the volume density and number of lesions increased with age, however, the affected region was restricted to the posteroventral cochlear nucleus and adjacent portions of the dorsal cochlear nucleus, interstitial nucleus, and posterior anteroventral cochlear nucleus. Lesions were also noted in a separate locus in the auditory nerve trunk associated with the acoustic nerve nucleus. The fusiform and molecular layers of the dorsal cochlear nucleus were spared at all ages observed. The spherical cell region of the arteroventral cochlear nucleus was also largely spared. A good correlation was observed between the cumulative input from the auditory nerve fibers caused by the ambient acoustic environment acting over the life of the animal and the number of lesions in tonotopic subdivisions of the cochlear nuclei. The earliest microcysts formed in regions receiving auditory nerve fibers most strongly stimulated by the ambient noise. Thereafter, short exposures to higher levels of input or long exposures to lower levels of input were quantitatively equivalent in producing microcystic lesions.

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.

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.

Localization of type II, IX and V collagen in the inner ear.

Types II and IX collagen are traditionally considered cartilage collagens; however, within the inner ear, types II and IX collagen have a more diverse distribution. In the adult gerbil, type II collagen is the major fibrillar component. In the otic capsule it is present surrounding the osteocytes embedded and branching in the periosteal layer, in the cartilaginous rests of the enchondral layer, and in the endosteal layer bordering the membranous labyrinth. In the regions of the sensory cells, type II collagen is found in the osseous spiral lamina, the connective tissue of the spiral limbus, the subepithelial tissue of the maculae in the vestibule and the cristae in the ampullae, and in the spiral ligament. It is present in the non-cartilaginous and acellular structures of the tectorial membrane over the cochlear hair cells and the vestibular membrane lining the semicircular canals. Type IX collagen, when present, in all cases co-localizes with type II collagen but is found in more limited regions. It is found only in the cartilaginous rests of the enchondral bone, the tectorial membrane and the vestibular membrane. Type V-like collagen, a connective tissue collagen, is found to have a complementary localization to types II and IX collagen within the interstitial bone of the otic capsule, the osseous spiral lamina and the tectorial membrane, but it is absent from the vestibular membrane. This report is the first documenting the co-localization of types II and IX collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

[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.

Characterization of inner ear sensory hair cells after rapid-freezing and freeze-substitution.

In order to determine the ultrastructural organization of normal cells and to understand better the anatomical substrates for outer hair cell motility, cryofixation was performed on the sensory epithelium of the inner ear of guinea pigs prior to substitution of frozen water with organic solvents containing chemical fixatives. In this way cells would not be altered by the direct application of the chemicals commonly used for preservation, which are also known to cause fixation-induced shape changes in outer hair cells. Following rapid freezing and freeze-substitution, preservation of cells within the isolated sensory epithelium containing the organ of Corti was similar to that seen in conventionally fixed cells. However, in rapidly frozen and freeze-substituted outer hair cells the cytoplasm and the cellular membranes differed from those seen in conventionally fixed preparations. The cytoplasmic matrix was densely packed with filaments and stained homogeneously, suggesting better preservation of the cytoskeleton and less extraction of the soluble ground substance. Cell membranes were smooth, indicating that fixation-induced shape changes and shrinkage had been avoided. The subsurface cisternal system of intracellular membranes lining the lateral wall of the outer hair cells was composed of continuous, tightly packed, parallel rows of membranous lamellae. Thus rapid-freezing and freeze-substitution are important techniques by which structural alterations correlated with outer hair cell motility can be separated from fixation-induced cell shape changes.