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Spiral ligament fibrocytes of the cochlea play homoeostatic roles in hearing and their degeneration contributes to hearing loss. Culturing fibrocytes in vitro provides a way to evaluate their functional characteristics and study possible therapies for hearing loss. We investigated whether in vivo characteristics of fibrocytes could be recapitulated in vitro by modifying the culture substrates and carried out proof of concept studies for potential transplantation of culture cells into the inner ear. Fibrocytes cultured from 4 to 5-week old CD/1 mice were grown on 2D substrates coated with collagen I, II, V or IX and, after harvesting, onto or into 3D substrates (hydrogels) of collagen I alone or mixed collagen I and II at a 1:1 ratio. We also assessed magnetic nanoparticle (MNP) uptake. Cell counts, immunohistochemical and ultrastructural studies showed that fibrocytes grown on 2D substrates proliferated, formed both small spindle-shaped and large flat cells that avidly took up MNPs. Of the different collagen coatings, only collagen II had an effect, causing a reduced size of the larger cells. On hydrogels, the cells were plump/rounded with extended processes, resembling native cells. They formed networks over the surface and became incorporated into the gel. In all culture formats, the majority co-expressed caldesmon, aquaporin 1, S-100 and sodium potassium ATPase, indicating a mixed or uncharacterised phenotype. Time-course experiments showed a decrease to ∼50% of the starting population by 4d after seeding on collagen I hydrogels, but better survival (∼60%) was found on collagen I + II gels, whilst TEM revealed the presence of apoptotic cells. Cells grown within gels additionally showed necrosis. These results demonstrate that fibrocytes grown in 3D recapitulate in vivo morphology of native fibrocytes, but have poorer survival, compared with 2D. Therefore hydrogel cultures could be used to study fibrocyte function and might also offer avenues for cell-replacement therapies, but need more optimization for therapeutic use. Fibrocyte function could be modified using MNPs in combination, for example, with gene transfection.

Transmembrane channel like protein 1 (TMC1) is likely to be a pore-forming subunit of the transduction channel of cochlear hair cells that is mechanically gated by tension on tip links in the stereocilia bundle. To localise TMC1 precisely, we labelled mice cochleae of different ages using custom-made polyclonal antibodies to TMC1 for light and transmission electron microscopy (TEM). Immunofluorescence revealed stereocilia labelling at P9 but not at P3 in apical hair cells. Immunogold labelling for TEM confirmed that labelling was absent at P3, and showed weak labelling at P6 with no stereocilia tip labelling, increasing at P9, with specific tip labelling on shorter stereocilia and some throughout the bundle. At P12 and P21, labelling was refined mostly to stereocilia tips. Quantification showed that labelling overall reached maximum by P12, labelling per tip was relatively constant from P9 to P21, but percent tips labelled was reduced from 16% to 8%. Tmc1−/− showed no labelling. Thus TMC1 occurs at the lower end of the tip link, supporting its presence in the MET complex and likely the channel. Tip localisation from P9 onwards coincides with lipoma HMGIC fusion partner-like 5 (LHFPL5), a protein that may be involved in acquiring/maintaining TMC1 localisation.

Sound stimuli elicit movement of the stereocilia that make up the hair bundle of cochlear hair cells, putting tension on the tip links connecting the stereocilia and thereby opening mechanotransducer (MT) channels. Tmc1 and Tmc2, two members of the transmembrane channel–like family, are necessary for mechanotransduction. To assess their precise role, we recorded MT currents elicited by hair bundle deflections in mice with null mutations of Tmc1, Tmc2, or both. During the first postnatal week, we observed a normal MT current in hair cells lacking Tmc1 or Tmc2; however, in the absence of both isoforms, we recorded a large MT current that was phase-shifted 180°, being evoked by displacements of the hair bundle away from its tallest edge rather than toward it as in wild-type hair cells. The anomalous MT current in hair cells lacking Tmc1 and Tmc2 was blocked by FM1-43, dihydrostreptomycin, and extracellular Ca2+ at concentrations similar to those that blocked wild type. MT channels in the double knockouts carried Ca2+ with a lower permeability than wild-type or single mutants. The MT current in double knockouts persisted during exposure to submicromolar Ca2+, even though this treatment destroyed the tip links. We conclude that the Tmc isoforms do not themselves constitute the MT channel but are essential for targeting and interaction with the tip link. Changes in the MT conductance and Ca2+ permeability observed in the absence of Tmc1 mutants may stem from loss of interaction with protein partners in the transduction complex.

Calcium is tightly regulated in cochlear outer hair cells (OHCs). It enters mainly via mechanotransducer (MT) channels and is extruded by the PMCA2 isoform of the plasma membrane calcium ATPase, mutations in which cause hearing loss. To assess how pump expression matches the demands of Ca2+ homeostasis, the distribution of PMCA2 at different cochlear locations during development was quantified using immunofluorescence and post-embedding immunogold labeling. The PMCA2 isoform was confined to stereociliary bundles, first appearing at the base of the cochlea around post-natal day 0 (P0) followed by the middle and then the apex by P3, and was unchanged after P8. The developmental appearance matches maturation of the MT channels in rat OHCs. High-resolution immunogold labeling in adult rats showed PMCA2 was distributed along the membranes of all three rows of OHC stereocilia at similar densities and at about a quarter the density in IHC stereocilia. The difference between OHCs and inner hair cells (IHCs) is similar to the ratio of their MT channel resting open probabilities. Gold particle counts revealed no difference in PMCA2 density between low- and high-frequency OHC bundles despite larger MT currents in high-frequency OHCs. The PMCA2 density in OHC stereocilia was determined in low- and high-frequency regions from calibration of immunogold particle counts as 2200/μm2 from which an extrusion rate of ~200 ions·s−1 per pump was inferred. The limited ability of PMCA2 to extrude the Ca2+ load through MT channels may constitute a major cause of OHC vulnerability and high-frequency hearing loss.

Spiral ligament fibrocytes function in cochlear homeostasis, maintaining the endocochlear potential by participating in potassium recycling, and fibrocyte degeneration contributes to hearing loss. Their superficial location makes them amenable to replacement by cellular transplantation. Fibrocyte cultures offer one source of transplantable cells, but determining what fibrocyte types they contain and what phenotype transplanted cells may adopt is problematic. Here, we use immunogold electron microscopy to assess the relative expression of markers in native fibrocytes of the CD/1 mouse spiral ligament. Caldesmon and aquaporin 1 are expressed more in type III fibrocytes than any other type. S-100 is strongly expressed in types I, II, and V fibrocytes, and α1Na,K-ATPase is expressed strongly only in types II and V. By combining caldesmon or aquaporin 1 with S-100 and α1Na,K-ATPase, a ratiometric analysis of immunogold density distinguishes all except type II and type V fibrocytes. Other putative markers (creatine kinase BB and connective tissue growth factor) did not provide additional useful analytical attributes. By labeling serial sections or by double or triple labeling with combinations of three antibodies, this technique could be used to distinguish all except type II and type V fibrocytes in culture or after cellular transplantation into the lateral wall.

Presbycusis (age-related hearing loss) can result from various cochlear pathologies. We have studied the time course of degeneration in a mouse that shows accelerated presbycusis, the CD/1 mouse, as a possible model to investigate stem-cell strategies to prevent or ameliorate presbycusic changes. CD/1 mice from 0 to 72 weeks old were examined by light and electron microscopy. Early pathological changes were detected in basal turn spiral ligament fibrocytes and spiral ganglion, but the latter was variable as both satellite cells and neurons were normal in some cochleae. Light microscopic counts in the spiral ligament of 20-week-old mice revealed that of the five main types (types I-V), only type V fibrocytes showed no reduction in numbers compared with 3-week-old animals, and type IV showed the greatest losses. However, all types of fibrocyte showed subtle damage when examined using electron microscopy, in the form of swollen mitochondria, as early as 2 weeks. The extent of mitochondrial damage showed a degree of correspondence with the light microscopic pattern of fibrocyte loss in that types III and IV fibrocytes had the most abnormal mitochondria and type V the least, especially at early stages. By 10-15 weeks, ultrastructural features of fibrocyte damage were similar to longer term changes reported in gerbils. Stria vascularis, spiral ganglion and hair cells showed few consistent early signs of damage but became increasingly affected, lagging behind the fibrocyte damage. Our data suggest that fibrocyte pathology may precede other presbycusic changes; breakdown of homeostatic mechanisms to which they contribute may cause the subsequent degeneration of the hair cells. Overall, there were many similarities to presbycusic changes in other rodents and humans. Therefore, the features of accelerated aging in this mouse make it a suitable model for rapidly assessing possible strategies to prevent or ameliorate presbycusic changes.

Outer hair cells (OHCs) of the mammalian cochlea besides being sensory receptors also generate force to amplify sound-induced displacements of the basilar membrane thus enhancing auditory sensitivity and frequency selectivity. This force generation is attributable to voltage-dependent contractility of the OHCs underpinned by the motile protein, prestin. Prestin is located in the basolateral wall of OHCs and is thought to alter its conformation in response to changes in membrane potential. The precise ultrastructural distribution of prestin was determined using post-embedding immunogold labelling and the density of the labelling was compared in low and high frequency regions of the cochlea. The labelling was confined to the basolateral plasma membrane in hearing rats but declined towards the base of the cells below the nucleus. In pre-hearing animals, prestin labelling was lower in the membrane and also occurred in the cytoplasm, presumably reflecting its production during development. The densities of labelling in low-frequency and high-frequency regions of the cochlea were similar. Non-linear capacitance, thought to reflect charge movements during conformational changes in prestin, was measured in OHCs in isolated cochlear coils of hearing animals. OHC non-linear capacitance in the same regions assayed in the immunolabelling was also similar in both apex and base, with charge densities of 10,000 /µm2 expressed relative to the lateral membrane area. The results suggest that prestin density and by implication force production, is similar in low-frequency and high-frequency OHCs.

Calcium buffers are important for shaping and localizing cytoplasmic Ca2+transients in neurons. We measured the concentrations of the four main calcium-buffering proteins (calbindin-D28k, calretinin, parvalbumin-α, and parvalbumin-β) in rat cochlear hair cells in which Ca2+signaling is a central element of fast transduction and synaptic transmission. The proteins were quantified by calibrating immunogold tissue counts against gels containing known amounts of each protein, and the method was verified by application to Purkinje cells in which independent estimates exist for some of the protein concentrations. The results showed that, in animals with fully developed hearing, inner hair cells had\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\frac{1}{10}\) \end{document}of the proteinaceous calcium buffer of outer hair cells in which the cell body contained parvalbumin-β (oncomodulin) and calbindin-D28k at levels equivalent to 5 mmcalcium-binding sites. Both proteins were partially excluded from the hair bundles, which may permit fast unbuffered Ca2+regulation of the mechanotransducer channels. The sum of the calcium buffer concentrations decreased in inner hair cells and increased in outer hair cells as the cells developed their adult properties during cochlear maturation. The results suggest that Ca2+has distinct roles in the two types of hair cell, reflecting their different functions in auditory transduction. Ca2+is used in inner hair cells primarily for fast phase-locked synaptic transmission, whereas Ca2+may be involved in regulating the motor capability underlying cochlear amplification of the outer hair cell. The high concentration of calcium buffer in outer hair cells, similar only to skeletal muscle, may protect against deleterious consequences of Ca2+loading after acoustic overstimulation.

Spherical bushy neurons in the anteroventral cochlear nucleus receive glutamatergic primary terminals from the cochlear nerve and terminals of noncochlear (i.e. nonprimary) origin, many of which colocalize gamma-aminobutyric acid (GABA) and glycine. Here the relationship between GABA and glycine in these terminals has been investigated using postembedding immunogold labelling. A significant negative correlation was found between the density of terminal labelling for GABA and for glycine in four guinea pigs. Terminals could be divided into three categories, GABA-only, glycine-only, or colocalizing depending on whether they had a significantly higher labelling density for either amino acid than the primary terminals. The overall labelling density in all four animals was significantly greater for GABA in GABA-only terminals than colocalizing ones but similar for glycine in both. Within the terminals, the labelling density over synaptic vesicles, nonvesicular regions of cytoplasm and mitochondria was also investigated. No significant difference was detected in the labelling density of vesicles compared with nonvesicular regions for either amino acid. However, a significant difference was found between the overall labelling density over mitochondria and nonvesicular regions for both. There was also significantly more mitochondrial GABA labelling in GABA-only terminals compared to colocalizing terminals but mitochondrial glycine labelling was similar in glycine-only and colocalizing terminals. Thus the level of GABA is higher in single than in colocalizing terminals, particularly in the mitochondria, but similar for glycine in both. It is possible therefore that the presence of glycine affects the level of GABA in the nonprimary terminals but that the presence of GABA does not affect the level of glycine.

The apices of the majority of cells of the organ of Corti are connected together by junctional complexes to form the reticular lamina, a barrier that prevents the mixing of endolymph and perilymph. These complexes include tight junctions, adherens junctions and desmosomes. Further information is required about the identity and distribution of the molecules involved in these connections if the function and organization of the reticular lamina are to be well understood. One major category of molecules occurring in adherens junctions and desmosomes, and involved in the maintenance of tissue integrity, is the cadherins. However, although cadherin has been identified in junctions between supporting cells in the adult mammalian organ of Corti at the light microscopic level, its ultrastructural distribution has not so far been described. A post-embedding immunogold labelling technique has therefore been used in conjunction with a monoclonal antibody to cadherin to investigate its ultrastructural distribution in the adult guinea-pig reticular lamina. Immunolabelling is observed in hair cell-supporting cell junctions and in supporting cell-supporting cell junctions. In addition, there is more labelling associated with inner hair cell-supporting cell junctions than with outer hair cell-supporting cell junctions. This may indicate that the junctions associated with the two types of hair cell have different functional properties.

The avian auditory papilla contains two classes of sensory receptor, tall hair cells (THCs) and short hair cells (SHCs), the latter analogous to mammalian outer hair cells with large efferent but sparse afferent innervation. Little is known about the tuning, transduction, or electrical properties of SHCs. To address this problem, we made patch-clamp recordings from hair cells in an isolated chicken basilar papilla preparation at 33°C. We found that SHCs are electrically tuned by a Ca2+-activated K+ current, their resonant frequency varying along the papilla in tandem with that of the THCs, which also exhibit electrical tuning. The tonotopic map for THCs was similar to maps previously described from auditory nerve fiber measurements. SHCs also possess an A-type K+ current, but electrical tuning was observed only at resting potentials positive to −45 mV, where the A current is inactivated. We predict that the resting potential in vivo is approximately −40 mV, depolarized by a standing inward current through mechanotransducer (MT) channels having a resting open probability of ∼0.26. The resting open probability stems from a low endolymphatic Ca2+ concentration (0.24 mM) and a high intracellular mobile Ca2+ buffer concentration, estimated from perforated-patch recordings as equivalent to 0.5 mM BAPTA. The high buffer concentration was confirmed by quantifying parvalbumin-3 and calbindin D-28K with calibrated postembedding immunogold labeling, demonstrating >1 mM calcium-binding sites. Both proteins displayed an apex-to-base gradient matching that in the MT current amplitude, which increased exponentially along the papilla. Stereociliary bundles also labeled heavily with antibodies against the Ca2+ pump isoform PMCA2a.

Calcium is tightly regulated in cochlear outer hair cells (OHCs). It enters mainly via mechanotransducer (MT) channels and is extruded by the plasma membrane calcium ATPase (PMCA)2 isoform of the PMCA, mutations in which cause hearing loss. To assess how pump expression matches the demands of Ca(2+) homeostasis, the distribution of PMCA2 at different cochlear locations during development was quantified using immunofluorescence and post-embedding immunogold labeling. The PMCA2 isoform was confined to stereociliary bundles, first appearing at the base of the cochlea around post-natal day (P)0 followed by the middle and then the apex by P3, and was unchanged after P8. The developmental appearance matched the maturation of the MT channels in rat OHCs. High-resolution immunogold labeling in adult rats showed that PMCA2 was distributed along the membranes of all three rows of OHC stereocilia at similar densities and at about a quarter of the density in inner hair cell stereocilia. The difference between OHCs and inner hair cells was similar to the ratio of their MT channel resting open probabilities. Gold particle counts revealed no difference in PMCA2 density between low- and high-frequency OHC bundles despite larger MT currents in high-frequency OHCs. The PMCA2 density in OHC stereocilia was determined in low- and high-frequency regions from calibration of immunogold particle counts as 2200/μm(2) from which an extrusion rate of ∼200 ions/s per pump was inferred. The limited ability of PMCA2 to extrude the Ca(2+) load through MT channels may constitute a major cause of OHC vulnerability and high-frequency hearing loss.

Outer hair cells (OHCs) of the mammalian cochlea besides being sensory receptors also generate force to amplify sound-induced displacements of the basilar membrane thus enhancing auditory sensitivity and frequency selectivity. This force generation is attributable to the voltage-dependent contractility of the OHCs underpinned by the motile protein, prestin. Prestin is located in the basolateral wall of OHCs and is thought to alter its conformation in response to changes in membrane potential. The precise ultrastructural distribution of prestin was determined using post-embedding immunogold labelling and the density of the labelling was compared in low-frequency and high-frequency regions of the cochlea. The labelling was confined to the basolateral plasma membrane in hearing rats but declined towards the base of the cells below the nucleus. In pre-hearing animals, prestin labelling was lower in the membrane and also occurred in the cytoplasm, presumably reflecting its production during development. The densities of labelling in low-frequency and high-frequency regions of the cochlea were similar. Non-linear capacitance, thought to reflect charge movements during conformational changes in prestin, was measured in OHCs in isolated cochlear coils of hearing animals. The OHC non-linear capacitance in the same regions assayed in the immunolabelling was also similar in both the apex and base, with charge densities of 10,000/microm(2) expressed relative to the lateral membrane area. The results suggest that prestin density, and by implication force production, is similar in low-frequency and high-frequency OHCs.

There are several subtypes of fibrocyte in the spiral ligament and spiral limbus of the cochlea that may contribute to fluid homeostasis. Immunocytochemical data suggest that these fibrocytes possess the glutamate–aspartate transporter, GLAST, as do supporting cells around the hair cells. However, functional glutamate uptake has not been demonstrated in fibrocytes. We used confocal and post-embedding immunogold electron microscopy to confirm that GLAST is expressed in adult fibrocytes of CD-1 mice with a relative expression: spiral limbus fibrocytes>type II>V>IV>I spiral ligament fibrocytes. Because they were sparsely present in most samples, type III fibrocytes were assessed only in one sample where their GLAST levels were similar to type I. Type II, type V and spiral limbus fibrocytes have many fine cellular processes that increase their surface area, those of the latter two coming into direct contact with perilymph, and type V fibrocytes contain the most glutamate. These data imply that glutamate uptake occurs in the fibrocytes. We assessed uptake of d -aspartate (a glutamate analogue) together with GLAST expression immunocytochemically and electrophysiologically. d -Aspartate accumulated into GLAST expressing fibrocytes in vitro and evoked currents blockable by the GLAST inhibitor d,l -threo-β-benzyloxyaspartate (TBOA), similar to those of supporting cells around inner hair cells. Currents were strongest in spiral limbus fibrocytes, progressively lower in type V and type II fibrocytes, and were negligible in type I fibrocytes in accordance with the relative expression levels of GLAST. We conclude that in addition to their known homeostatic functions, fibrocytes, in particular spiral limbus, type II and type V fibrocytes play a role in glutamate homeostasis in the cochlea.

The sensory bundle of vertebrate cochlear hair cells consists of actin-containing stereocilia that are thought to bend at their ankle during mechanical stimulation. Stereocilia have dense rootlets that extend through the ankle region to anchor them into the cuticular plate. Because this region may be important in bundle stiffness and durability during prolonged stimulation at high frequencies, we investigated the structure and dimensions of rootlets relative to the stereocilia in apical (low-frequency) and basal (high-frequency) regions of rodent cochleae using light and electron microscopy. Their composition was investigated using postembedding immunogold labeling of tropomyosin, spectrin, beta-actin, gamma-actin, espin, and prestin. The rootlets have a thick central core that widens at the ankle, and are embedded in a filamentous meshwork in the cuticular plate. Within a particular frequency region, rootlet length correlates with stereociliary height but between regions it changes disproportionately; apical stereocilia are, thus, approximately twice the height of basal stereocilia in equivalent rows, but rootlet lengths increase much less. Some rootlets contact the tight junctions that underlie the ends of the bundle. Rootlets contain spectrin, tropomyosin, and beta- and gamma-actin, but espin was not detected; spectrin is also evident near the apical and junctional membranes, whereas prestin is confined to the basolateral membrane below the junctions. These data suggest that rootlets strengthen the ankle region to provide durability and may contact with the lateral wall either to give additional anchoring of the stereocilia or to provide a route for interactions between the bundle and the lateral wall.

Sensory and supporting cells of the mammalian organ of Corti have cytoskeletons containing beta- and gamma-actin isoforms which have been described as having differing intracellular distributions in chick cochlear hair cells. Here, we have used post-embedding immunogold labelling for beta- and gamma-actin to investigate semiquantitatively how they are distributed in the guinea-pig cochlea and to compare different frequency locations. Amounts of beta-actin decrease and gamma-actin increase in the order, outer pillar cells, inner pillar cells, Deiters' cells and hair cells. There is also more beta-actin and less gamma-actin in outer pillar cells in higher than lower frequency regions. In hair cells, beta-actin is present in the cuticular plate but is more concentrated in the stereocilia, especially in the rootlets and towards the periphery of their shafts; labelling densities for gamma-actin differ less between these locations and it is the predominant isoform of the hair-cell lateral wall. Alignments of immunogold particles suggest beta-actin and gamma-actin form homomeric filaments. These data confirm differential distribution of these actin isoforms in the mammalian cochlea and reveal systematic differences between sensory and supporting cells. Increased expression of beta-actin in outer pillar cells towards the cochlear base may contribute to the greater stiffness of this region.

Hair cells of the inner ear contain high concentrations of calcium-binding proteins that limit calcium signals and prevent cross talk between different signaling pathways during auditory transduction. Using light microscope immunofluorescence and post-embedding immunogold labeling in the electron microscope, we characterized the distribution of three calcium-buffering proteins in the turtle cochlea. Both calbindin-D28k and parvalbumin-β were confined to hair cells in which they showed a similar distribution, whereas calretinin was present mainly in hair-cell nuclei but also occurred in supporting cells and nerve fibers. The hair-cell concentration of calbindin-D28k but not of parvalbumin-β increased from the low- to high-frequency end of the cochlea. Calibration against standards containing known amounts of calcium-buffering protein processed in the same fluid drop as the cochlear sections gave cytoplasmic concentrations of calbindin-D28k as 0.13–0.63 mmand parvalbumin-β as ∼0.25 mm, but calretinin was an order of magnitude less. Total amount of Ca2+-binding sites on the proteins is at least 1.0 mmin low-frequency hair cells and 3.0 mmin high-frequency cells. Reverse transcription-PCR showed that mRNA for all three proteins was expressed in turtle hair cells. We suggest that calbindin-D28k and parvalbumin-β may serve as endogenous mobile calcium buffers, but the predominantly nuclear location of calretinin argues for another role in calcium signaling. The results support conclusions from electrophysiological measurements that millimolar concentrations of endogenous calcium buffers are present in turtle hair cells. Parvalbumin-β was also found in both inner and outer hair cells of the guinea pig cochlea.

Double labeling is used for localizing two antigens simultaneously in the same tissue. We have used two approaches to post-embedding immunogold labeling to investigate whether nerve terminals in the guinea-pig anteroventral cochlear nucleus (AVCN) that contain gamma-aminobutyric acid (GABA) or glycine are capable of retrieving the other amino acid as part of an investigation of colocalization of these putative neurotransmitters. For this, vibroslices of perfusion-fixed brain stem were freeze-substituted and embedded in the low temperature resin, Lowicryl HM20. Simultaneous labeling of ultrathin sections was then performed with a mixture of a rabbit primary antibody to GABA and a guinea-pig primary antibody to the glycine transporter, GLYT2, followed by labeling with a mixture of secondary antibodies (goat anti-rabbit IgG-30 nm gold, goat anti-guinea pig IgG-15 nm gold). This approach indicated that GLYT2 occurs in the plasma membrane of some terminals that contain GABA. The other approach involved sequential labeling of ultrathin sections with a rabbit primary antibody to the GABA transporter, GAT1, followed by an anti-rabbit secondary antibody conjugated to 15-nm gold particles. Sections were then treated with paraformaldehyde vapor to denature any free anti-IgG binding sites on the first antibody, and labeled with a primary antibody to glycine also raised in rabbit followed by an anti-rabbit secondary antibody conjugated to 30-nm gold particles. This approach indicated that GAT1 occurs in the plasma membrane of some terminals that contain glycine. Thus, these techniques can be used to localize heat-labile multiple antigens in the same tissue.

The majority of terminals contacting spherical bushy cell bodies in the guinea-pig anteroventral cochlear nucleus contain GABA, glycine or both (colocalizing). Double labeling with antibodies to each amino acid and the plasma membrane transporter for the other was performed using different sizes of gold particles. The transporter for GABA occurs in the plasma membranes of some terminals containing glycine and vice versa suggesting that colocalizing terminals can retrieve both amino acids.

Spectrin is a cytoskeletal protein found in the cortex of many cell types. It is known to occur in cochlear outer hair cells (OHCs) with previous immunoelectron microscopical studies showing that it is located in the cuticular plate and the cortical lattice. The latter is a network of filaments associated with the lateral plasma membrane that is thought to play a role in OHC motility. Spectrin has also been found in inner hair cells (IHCs) and supporting cells using immunofluorescent techniques, but its ultrastructural distribution in these cells has not yet been described. This has, therefore, been investigated using a monoclonal antibody to alpha-spectrin in conjunction with pre- and post-embedding immunogold labelling for transmission electron microscopy. Labelling was found in a meshwork of filaments beneath the plasma membranes of both IHCs and supporting cells and, in pillar cells, close to microtubule/microfilament arrays. It was also found in association with the stereocilia of OHCs and IHCs and, as expected, in the cortical lattice and cuticular plate of OHCs. Thus, spectrin is a general component of cytoskeletal structures involved in maintaining the specialised cell shapes in the organ of Corti and may contribute to the mechanical properties of all the cell types examined.

The localisation and distribution of the cholinergic and serotoninergic components of the nervous system in the plerocercoid, adult and free proglottis stages of the tetraphyllidean tapewormTrilocularia acanthiaevulgaris were determined by enzyme histochemical and immunocytochemical techniques. The central nerve ring (CNR) in the scolex contains two lateral ganglia and gives rise to five pairs of longitudinal nerve cords (LNC's; three lateral, two median). The nerve cords run posteriorly throughout the bodies of the plerocercoid and adult worms and the free proglottis. Nerves from the CNR and accessory lateral LNC's pass to the bothridia, where they give rise to extensive nerve plexuses. As the individual proglottides develop along the strobila, a small nerve ring forms at the anterior end of each proglottis; within the nerve ring, distinct bilateral ganglia develop prior to the release of the proglottis. All ten LNC's are present in the free proglottis. The genital atrium and cirrus sac are innervated by cholinergic and serotoninergic elements. The cholinergic nervous system predominates in the CNS within the scolex, whereas there is a larger population of 5-HT-immunoreactive nerve cells associated with the LNC's and segmental ganglia along the strobila and within the free proglottis.

The early (ovarian) stages of oogenesis in Trilocularia acanthiaevulgaris have been studied by light microscope histochemistry and transmission electron microscopy. The process proceeds as far as meiotic prophase in the primary oocyte. The oogonia and early immature primary oocytes occupy the anterior and outer edges of the ovary and are typically undifferentiated cells, showing a high nucleo-cytoplasmic ratio. The scant cytoplasm is packed with free ribosomes and contains a small number of mitochondria and a few short strands of granular endoplasmic reticulum (GER). The oogonia undergo a number of mitotic divisions, marked by the presence of centrioles. The immature primary oocytes enter meiotic prophase, as evidenced by the appearance of nuclear synaptonemal complexes. The maturing primary oocytes, which occupy the inner, central region of the ovary, undergo a growth phase that is accompanied by an increased nucleolar volume and export of RNA to the cytoplasm via nuclear pores, and by an increase in the number of mitochondria. A GER network develops and, together with the Golgi complexes, is involved in the production of a small number of cortical granules. The GER often takes the form of concentric cisternae. Numerous lipid droplets are also present in the cytoplasm. The mature primary oocytes predominate in the posterior region of the ovary, near the oviduct. They represent a resting phase in development, in which cellular activity is minimal. Lipid droplets are abundant and the cortical granules remain in more central regions of the cell and do not migrate to the periphery of the cell. The ovary contains a second, non-germinal type of cell, the follicle cell. The cell body is smaller in size than the oogonia, and cytoplasmic processes from it ramify around the periphery of the ovary. The different cell types within the ovary are embedded in a cytoplasmic matrix that contains a number of organelles.

Spermatogenesis and the structure of the mature spermatozoon of Trilocularia acanthiaevulgaris have been studied by light microscopic histochemistry and transmission and scanning electron microscopy. The primary spermatogonia occupy the periphery of the testis and undergo four mitotic divisions; incomplete cytokinesis results in two secondary, four tertiary and eight quaternary spermatogonia and sixteen primary spermatocytes. The quaternary spermatogonia and successive stages develop in a plasmodial rosette with their nuclei at the periphery and connected to the cytophore by cytoplasmic bridges. The primary spermatocytes undergo two meiotic divisions, resulting in 64 spermatids that develop into spermatozoa. Intranuclear synaptonemal complexes in primary spermatocytes confirm the first meiotic division. The onset of spermiogenesis is marked by an elongation of the spermatid nucleus and the appearance of "arching membrane-bound clefts" (after Rosario 1964) that delimit the differentiation zone. The latter contains a pair of axonemes with their associated basal bodies and rootlets and, as it extends, the nucleus migrates into it. At first, the axonemes grow at right angles to the extension, but during development they rotate through 90 degrees and come to fuse with the cytoplasmic extension. The mature spermatozoon has a broad head region, a middle piece and a narrow tail region. It consists of a highly coiled nucleus, paired axonemes of the 9 + 1 pattern typical of platyhelminths, one of which extends the full length of the tail and the other ends part-way along the tail, particles of beta-glycogen and peripheral microtubules. A crested body spirals around the outside of the sperm body in the tail region. T. acanthiaevulgaris is placed in context within the phylogenetic schemes for cestodes based on sperm ultrastructure.