Somatostatin receptor types 1 and 2 in the developing mammalian cochlea.

The neuropeptide somatostatin (SST) exerts several important physiological actions in the adult central nervous system through interactions with membrane-bound receptors. Transient expression of SST and its receptors has been described in several brain areas during early ontogeny. It is therefore believed that SST may play a role in neural maturation. The present study provides the first evidence for the developmental expression of SST receptors in the mammalian cochlea, emphasizing their possible roles in cochlear maturation. In the developing mouse cochlea, cells immunoreactive to somatostatin receptor 1 (SSTR1) and somatostatin receptor 2 (SSTR2) were located in the embryonic cochlear duct on Kolliker's organ as early as embryonic day (E) 14 (E14). At E17, the expression of both receptors was high and already located at the hair cells and supporting cells along the length of the cochlear duct, which have become arranged into the characteristic pattern for the organ of Corti (OC) at this stage. At birth, SSTR1- and SSTR2-containing cells were only localized in the OC. In general, immunoreactivity for both receptors increased in the mouse cochlea from postnatal day (P) 0 (P0) to P10; the majority of immunostained cells were inner hair cells, outer hair cells, and supporting cells. Finally, a peak in the mRNA and protein expression of both receptors is present near the time when they respond to physiological hearing (i.e., hearing of airborne sound) at P14. At P21, SSTR1 and SSTR2 levels decrease dramatically. A similar developmental pattern was observed for SSTR1 and SSTR2 mRNA, suggesting that the expression of the SSTR1 and SSTR2 genes is controlled at the transcriptional level throughout development. In addition, we observed reduced levels of phospho-Akt and total Akt in SSTR1 knockout and SSTR1/SSTR2 double-knockout mice compared with wild-type mice. We know from previous studies that Akt is involved in hair cell survival. Taken together, the dynamic nature of SSTR1 and SSTR2 expression at a time of major developmental changes in the cochlea suggests that SSTR1 and SSTR2 (and possibly other members of this family) are involved in the maturation of the mammalian cochlea.

Activin potentiates proliferation in mature avian auditory sensory epithelium.

Humans and other mammals are highly susceptible to permanent hearing and balance deficits due to an inability to regenerate sensory hair cells lost to inner ear trauma. In contrast, nonmammalian vertebrates, such as birds, robustly regenerate replacement hair cells and restore hearing and balance functions to near-normal levels. There is considerable interest in understanding the cellular mechanisms responsible for this difference in regenerative capacity. Here we report on involvement of the TGFbeta superfamily type II activin receptors, Acvr2a and Acvr2b, in regulating proliferation in mature avian auditory sensory epithelium. Cultured, posthatch avian auditory sensory epithelium treated with Acvr2a and Acvr2b inhibitors shows decreased proliferation of support cells, the cell type that gives rise to new hair cells. Conversely, addition of activin A, an Acvr2a/b ligand, potentiates support cell proliferation. Neither treatment (inhibitor or ligand) affected hair cell survival, suggesting a specific effect of Acvr2a/b signaling on support cell mitogenicity. Using immunocytochemistry, Acvr2a, Acvr2b, and downstream Smad effector proteins were differentially localized in avian and mammalian auditory sensory epithelia. Collectively, these data suggest that signaling through Acvr2a/b promotes support cell proliferation in mature avian auditory sensory epithelium and that this signaling pathway may be incomplete, or actively blocked, in the adult mammalian ear.

Localization of megalin in rat vestibular dark cells and endolymphatic sac epithelial cells.

CONCLUSION: Megalin immunoreactivity was observed in kidney proximal tubule cells, vestibular dark cells, and epithelial cells of the endolymphatic sac. Endocytic mechanisms appear to differ between the endolymphatic sac and proximal tubule cells. We speculate that megalin is secreted by a certain type of cell into the endolymphatic space, and is then absorbed from the endolymphatic space by another type of cell to maintain endolymphatic sac homeostasis. OBJECTIVES: We previously detected megalin immunoreactivity in the rat cochlear duct. Megalin may be involved in endocytosis in the vestibular organ and endolymphatic sac. To examine this possibility, we extended our immunocytochemical investigation to the rat inner ear cells with special attention to vestibular dark cells and endolymphatic sac. MATERIALS AND METHODS: We observed immunoreactivity of megalin under light and electron microscopy. The primary antibody was rabbit polyclonal antibody that had been raised against rat immunoaffinity-purified megalin. RESULTS: The luminal membrane and subapical area of dark cells in the semicircular canal were immunolabeled. The stainable substance in the endolymphatic space was strongly stained. The cytoplasm of epithelial cells was also stained in various patterns.

Cellular growth and rearrangement during the development of the mammalian organ of Corti.

The sensory epithelium of the mammalian cochlea, the organ of Corti, is comprised of ordered rows of cells, including inner and outer hair cells. Recent results suggest that physical changes in the overall size and shape of the cochlear duct, including possible convergence and extension, could play a role in the development of this pattern. To examine this hypothesis, changes in cell size and distribution were determined for different regions of the cochlea duct during embryonic development. In addition, changes in the spatial distribution of sensory precursor cells were determined at different developmental time points based on expression of p27kip1. Unique changes in luminal surface area, cell density, and number of cell contacts were observed for each region of the duct. Moreover, the spatial distribution of p27kip1-positive cells changed from short and broad early in development, to long and narrow. These results are consistent with the hypothesis that convergence and extension plays a role in cellular patterning within the organ of Corti.

K+ and Na+ absorption by outer sulcus epithelial cells.

Transduction of sound into nerve impulses by hair cells depends on modulation of a current carried primarily by K+ into the cell across apical transduction channels that are permeable to cations. The cochlear function thus depends on active secretion of K+ accompanied by absorption of Na+ by epithelial cells enclosing the cochlear duct. The para-sensory cells which participate in the absorption of Na+ (down to the uniquely low level of 1 mM) were previously unidentified and the existence of a para-sensory pathway which actively absorbs K+ was previously unknown. A relative short circuit current (Isc,probe, measured as the extracellular current density with a vibrating electrode) was directed into the apical side of the outer sulcus epithelium, decreased by ouabain (1 mM), an inhibitor of Na+, K(+)-ATPase, and found to depend on bath Na+ and K+ but on neither Ca2+ nor Cl-. Isc,probe was shown to be an active current by its sensitivity to ouabain. On-cell patch clamp recordings of the apical membrane of outer sulcus cells displayed a channel activity, which carried inward currents under conditions identical to those used to measure Isc,probe. Both Isc,probe and non-selective cation channels (27.4+/-0.6 ps, n = 22) in excised outside-out patches from the apical membrane were inhibited by Gd3+ (1 mM). Ics,prob was also inhibited by 5 mM lidocaine, 1 mM quinine and 500 microM amiloride but not by 10 microM amiloride. These results demonstrate that outer sulcus epithelial cells contribute to the homeostasis of endolymph by actively absorbing Na+ and K+. An entry pathway in the apical membrane was shown to be through non-selective cation channels that were sensitive to Gd3+.

Structural maturation of the interface region between the stria vascularis and spiral ligament in the neonatal rat cochlea.

The ultrastructural morphology of the interface region between the stria vascularis (SV) and spiral ligament (SL) was examined in the neonatal rat cochlea via transmission electron microscopy. At postnatal day (PND) 3, morphology of both basal cells and fibrocytes was simple and immature. Only a small number of fibrocytes was observed in the SL. Intercellular junctions between basal cells and fibrocytes, and between adjacent fibrocytes, were few. At PND 7, the number of fibrocytes increased, and more organelles appeared within their cytoplasm. From PND 11 to 14, nuclei of the basal cells appeared to be more spindle-shaped and contained more heterochromatin. The cytoplasm of the fibrocytes was pale, and a greater number of cytoplasmic vesicles and mitochondria emerged. More intercellular junctions were observed between basal cells and fibrocytes at the interface region and between fibrocytes in the SL. By PND 21, the morphology of basal cells and fibrocytes and their intercellular junctions appeared to be adult-like. These morphological observations correlate with previous reports on the functional maturation of the developing rat cochlea.

Differentiation of the rat stria vascularis.

The differentiation of the rat stria vascularis (SV) was investigated by light- and electron microscopy and by immunocytochemistry. Loss of the basal lamina at the epithelial-mesenchymal interface of SVs as indicated by immunoreactions of laminin and fibronectin induces the formation of vascular feet by basal infoldings of the marginal cells (MCs), and the development of the strial capillaries (SCs) by mesenchymal cells in a manner of vasculogenesis is progressing at the same time. The production of fibronectin in the rough endoplasmic reticulum of mesenchymal cells and the involvement of this glycoprotein in a mechanical linkage between the vasoformative mesenchymal cells and endothelial ones of the SCs are indicated by immunocytochemistry. The plasma membrane of the marginal cells (MCs) begins to show immunoreactions of Na+.K+ ATPase at postnatal day 5 and is conjugated to each other by tight junctions at postnatal day 14. The apical tubules of the differentiating MCs do not seem to be involved in the endocytotic activity but are involved in the plasma membrane supply for the rapid differentiation.

Primary culture of vital marginal cells from cochlear explants of the stria vascularis.

Explants of the stria vascularis and spiral ligament were dissected from guinea pig cochleae and were successfully cultivated for several weeks. After 2 days, fibroblast-like cells of the spiral ligament covered the bottom of the cell culture dish around the explant. Marginal cells of the stria vascularis proliferated and grew on the luminal surface towards the border of the explant at a rate of 15 microns/day. At day 6 in culture the proliferating marginal cells reached the border of the explant and then advanced to the bottom of the cell-culture dish. There the marginal cells replaced fibroblast-like cells and built an epithelial hexagonal-shaped monolayer. Light microscopic and transmission electron microscopic investigations revealed that the cultured cells were viable and that typical morphological characteristics of marginal cells were preserved. Cultivation of these cells provides a unique model for studies of physiological properties of marginal cells of the stria vascularis.

Mesothelium of Reissner's membrane in guinea pigs: an electron microscopic study.

The mesothelial cells of Reissner's membrane in guinea pigs were found to be connected by junctional complexes. No cell discontinuities or gaps were observed by scanning or transmission electron microscopy. These results are not in accordance with previous studies. They were achieved by in vivo vascular perfusion fixation, handling of cochleae in protective specimen carriers, thiocarbohydrazide treatment and continuous dehydration. Findings in the present study indicate that the interepithelial space between the epithelial and mesothelial cell layers constitutes a specific compartment which must be considered when examining solute transport over Reissner's membrane.

Spiral prominence epithelium.

Recent studies have shown neuroendocrine-type cells in human spiral prominence epithelium. A light microscopic and ultrastructural study was made on the guinea pig spiral prominence epithelium to try and identify neuroendocrine cells. The study showed that such cells were not present in the guinea pig spiral prominence epithelium. The spiral prominence epithelium was characterized by uniformity of cellular staining with Toluidine blue and the ultrastructural appearance. The morphology of the spiral prominence epithelium was similar to that previously described. The absence of neuroendocrine cells in spiral prominence epithelium in the guinea pig is discussed in relation to species differences.

The developmental appearance of a major basilar papilla-specific protein in the chick.

The sensory epithelium of the chick cochlea, the basilar papilla, contains a major protein of approximately 23,000 daltons. This protein was as abundant as actin in the papilla, yet could not be found in significant quantities in any other cochlear tissue. The protein appeared at a time in development when other studies have shown that the chick embryo develops peripheral auditory competence. These observations suggest a role for this protein in cochlear function.