Bradley A. Schulte

The fine structure of spiral ligament cells relates to ion return to the stria and varies with place-frequency

Ultrastructural analysis of cells in the cochleas lateral wall was undertaken to investigate morophologic features relevant to the route of K+ cycling from organ of Corti (OC) to stria vascularis (StV) and to the question of a transcellular versus an extracellular path. The fine structure of outer sulcus cells (OSCs) evidenced their capacity for uptake of K+ from Claudius cells and from perilymph in inferior spiral ligament. Plasmalemmal amplification and mitochondrial density together with known content of Na,K-ATPase testified to activity of type II, IV and V fibrocytes in resorbing K+. Location and fine structure afforded a basis for distinguishing subtypes among the type I, II and IV cells. The type II, IV and V fibrocytes can be viewed as drawing K+ from surrounding perilymph and from OSCs and generating an intracellular downhill diffusion gradient for K+ flow through gap junctions to subtype Ib and Ia fibrocytes and strial basal cells. Pumping action enabled by extreme structural specialization of type II fibrocytes is considered to mediate K+ translocation across the interruption between the gap junction connected epithelial and gap junction connected fibrocyte systems and to explain ion flow directed toward StV through OSCs and fibrocytes despite their lack of polarity. The OSC bodies shrank, their root bundles expanded and the gap junction contact between OSCs and Claudius cells increased toward the base of the cochlea. Expanding root bundles and type I and IIb fibrocyte populations contrasted with shrinking OHCs and Deiters and tectal cells from the apex to the base of the cochlea. These differences indicated an increased magnitude and alternate route of K+ transport toward the StV in high as compared to low-frequency regions. The augmented K+ transport through spiral ligament in basal cochlea correlates with and provides a possible basis for the larger endocochlear potential in the base. The findings appear consistent with current flow extracellularly through scalae tympani and vestibuli and transcellularly through OC, OSCs and class I, II, IV and V fibrocytes.

Distribution of immunoreactive Na+,K+-ATPase in gerbil cochlea.

The distribution of Na+,K+-ATPase was mapped in cochleas of mature gerbils with normal hearing, using a specific and sensitive immunocytochemical method. Na+,K+-ATPase was abundant in the basolateral plasma membrane of marginal cells in the stria vascularis. Considerable levels of enzyme were also associated with the surfaces of spiral ganglion neurons and their central and peripheral processes. An unexpected finding was the detection of high levels of immunoreactive Na+,K+-ATPase in three different populations of cells lying in the inferior portion of the spiral ligament and at the medial and lateral border of the scala vestibuli just superior to the attachment of Reissners membrane. Cells in these areas shared the morphological characteristics of cells specialized for active transport but appeared to be nonpolarized, suggesting a uniform distribution of Na+,K+-ATPase over their entire plasmalemma. The presence of these three distinct cell populations in the cochlea of several mammalian species suggests that they play an important role in cochlear function, perhaps that of regulating the cation content of perilymph. The absence of discrete concentrations of Na+,K+-ATPase-rich cells in the perilymphatic connective tissue of the bird cochlea and the mammalian vestibular system suggests further that these cells may be involved with generating and maintaining the high endolymphatic potential unique to the mammalian cochlea.

Differentiation of inner ear fibrocytes according to their ion transport related activity

Fibrocytes in the lateral wall and limbus of the gerbil cochlea evidenced a capacity for ion transport activity by immunostaining for transport mediating enzymes including Na,K-ATPase, carbonic anhydrase (CA) and creatine kinase (CK). Fibrocytes of the spiral ligament unlike those in the suprastrial region and limbus decreased in abundance from base to apex. Spiral ligament fibrocytes at a given position along the cochlea varied in content of transport related enzymes, and on the basis of immunostaining, location and orientation, were classified into four types. Type I fibrocytes under the stria vascularis stained for CA isozymes II and III and CK isozyme BB. Type II fibrocytes under the outer sulcus and spiral prominence epithelium were found to contain only Na,K-ATPase. Type III fibrocytes lying adjacent to bone in the inferior region of the spiral ligament contained CA II and III and CK isozymes BB and MM. Type IV fibrocytes located more superficially in the inferior part of the spiral ligament stained variably for all the enzymes. Superficial fibrocytes in the suprastrial area disclosed Na,K-ATPase whereas the underlying fibrocytes stained for CA and CK. Limbal fibrocytes reacted with antisera to all the enzymes except CA III. Most fibrocytes in stromal plates beneath the vestibular systems neurosensory epithelium contained Na,K-ATPase and CA II but not CA III. These findings point to cooperativity in fluid and ion transport between epithelial cells and neighboring fibrocytes and demonstrate functional diversity of fibrocytes of the inner ear providing a basis for classifying those in the spiral ligament.

Lateral wall Na, K-ATPase and endocochlear potentials decline with age in quiet-reared gerbils

Changes in the integrity of cochlear ion transport systems with age were examined in gerbils raised for 5-38 months in a quiet environment. Ion transport function was assessed by light microscopic immunohistochemical staining for the enzyme, Na,K-ATPase and by measurement of the endocochlear potential (EP). Small foci of strial atrophy accompanied by loss of immunostaining for Na,K-ATPase were observed in the stria vascularis of the apical and basal turns as early as 5 months of age. Cochleas from 29-38 month-old gerbils showed a loss of immunostaining for Na,K-ATPase in the stria in most of the apical turn with the degeneration extending well into the middle turn in many of the oldest ears. The extent of strial atrophy and loss of immunoreactive Na,K-ATPase in the basal turn varied considerably among the oldest cochleas. Populations of lateral wall fibrocytes (type II fibrocytes) normally rich in Na,K-ATPase exhibited a corresponding decrease in enzyme content in regions of advanced strial atrophy. The volume of immunostained stria vascularis correlated well with the magnitude of the resting EP. The results demonstrate that lateral wall ion transport systems in the gerbil cochlea degenerate as a function of age. The findings also provide good evidence for a functional relationship between the stria vascularis and the Na,K-ATPase-rich type II fibrocytes in generating and maintaining the EP.

Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. II. Rat

SummarySalivary glands and pancreases from male rats were stained with a battery of ten different lectin-horseradish peroxidase conjugates. Qualitative and quantitative differences were observed in the content of terminal sugar residues in stored secretory glycoproteins in parenchymal cells of glands having a similar histological structure. Heterogeneity in the content of secretory glycoconjugates was also found between cells in the same exocrine glands, which were previously thought to be identical on the basis of classical morphological and histochemical staining studies. Similar differences were observed in the structure of glycoconjugates associated with the apical surface of epithelial cells lining glandular excretory ducts. Intercalated ducts presented a gland specific staining pattern different from that of the glandular secretory cell population, whereas striated duct and interlobular duct epithelial cells stained similarly in all major rat exocrine glands. A comparison of lectin binding patterns in identical histological sites in the mouse, reported in a companion paper, is provided, and the similarities and differences between these two rodent species are discussed. In addition to providing valuable information concerning the localization and structure of tissue complex carbohydrates, a comparison of staining in the same tissue sites with labelled lectins reported biochemically to have similar binding specificity has revealed interesting differences in the binding specificity of these macromolecules.

Histochemical localization of galactose-containing glycoconjugates in sensory neurons and their processes in the central and peripheral nervous system of the rat.

We studied the distribution of sugar residues in the oligosaccharide chains of complex carbohydrates in tissue sections of rat spinal cord, brainstem, and sensory ganglia using twelve lectin-horseradish peroxidase conjugates. Glycoconjugates containing terminal galactose residues were localized apparently in the Golgi apparatus in a population of predominantly small B-type neurons in spinal and trigeminal ganglia. Large A-type neurons rarely showed reactivity with galactose-binding lectins. A cells stained for glycoconjugates with N-glycosidically linked oligosaccharides and glycogen. The central and peripheral processes of the small neurons, mostly unmyelinated C fibers in sensory roots and spinal nerves, contained an abundance of glycoconjugates with terminal alpha-galactose residues. The central projections and terminals of small to medium-sized primary sensory neurons in the spinal and trigeminal ganglia were visualized in Lissauers tract and the substantia gelatinosa in the spinal cord, and in the spinal trigeminal tract and the nucleus trigeminus in the lower medulla with lectins specific for terminal alpha-galactose residues. In addition, fibers of the solitary system and the area postrema were reactive with these lectins. The peripheral and central nervous system elements with affinity for galactopyranosyl-specific lectins correspond in distribution with neuroanatomical regions thought to be involved in the transmission and relay of somatic and visceral afferent inputs such as pain and temperature. Such specific localization of a glycosubstance to a distinct subpopulation of neurons and their peripheral and central processes suggests that the particular glycoconjugate may be of physiological significance.

Immunohistochemical Localization of the Na-K-Cl Co-transporter (NKCC1) in the Gerbil Inner Ear:

We mapped the cellular and subcellular distribution of the Na-K-Cl co-transporter (NKCC) in the adult gerbil inner ear by immunostaining with a monoclonal antibody (MAb T4) generated against human colon NKCC. Heavy immunolabeling was seen in the basolateral plasma membrane of marginal cells in the stria vascularis and dark cells in the vestibular system. Subpopulations of fibrocytes in the cochlear spiral ligament and limbus and underlying the vestibular neurosensory epithelium also stained with moderate to strong intensity, apparently along their entire plasmalemma. Because MAb T4 recognizes both the basolateral secretory (NKCC1) and the apical absorptive (NKCC2) isoforms of the co-transporter, we employed reverse transcription and the polymerase chain reaction (RT-PCR) to explore isoform diversity in inner ear tissues. Using NKCC1 and NKCC2 isoform-specific PCR primers based on mouse and human sequences, only transcripts for NKCC1 were detected in the gerbil inner ear. The presence of abundant NKCC1 in the basolateral plasmalemma of strial marginal and vestibular dark cells confirms conclusions drawn from pharmacological and physiological data. The co-expression of NKCC1 and Na,K-ATPase in highly specialized subpopulations of cochlear and vestibular fibrocytes provides further evidence for their role in recycling K+ leaked or effluxed through hair cells into perilymph back to endolymph, as postulated in current models of inner ear ion homeostasis.

Evidence for a medial K+ recycling pathway from inner hair cells

K+ effluxed from outer hair cells and their nerves is thought to flow laterally to strial marginal cells for recycling into scala media. Observations reported here provide evidence that K+ effluxed from inner hair cells and inner radial nerves travels medially through border cells, inner sulcus cells (ISCs), limbal fibrocytes and interdental cells (IDCs) for return to endolymph. Morphologic features of ISCs in the medial route resembled those of Hensen and Claudius cells in the lateral indicating an ion transport role for ISCs like that of Hensen and Claudius cells. Na,K-ATPase in plasmalemma of IDCs testified to their capacity to resorb and transport K+ through their known gap junctions. IDCs were differentiated into three subgroups. The most lateral IDCs formed short and long columns. Long columns contacted the medialmost ISC inferiorly and the undersurface of the tectorial membrane superiorly providing thereby a potential transcellular route for K+ transit from ISCs to endolymph. Short columns faced inner sulcus below and tectorial membrane above and accordingly possessed cells with opposite polarity at the bottom and top of the column. Short columns thus appeared situated to resorb electrolytes from limbal stroma for release into inner sulcus and beneath tectorial membrane at opposite ends of the column. The central IDCs were positioned for resorbing and transporting K+ effluxing from the Na,K-ATPase-rich stellate fibrocytes which spread toward the IDCs from near the inner sulcus. The most medial IDCs lined cuplike invaginations near the attachment of Reissners membrane and lay apposed to light fibrocytes located between supralimbal fibrocytes and the medial IDCs. Content of Na,K-ATPase and position in the K+ transport route likened the limbal stellate fibrocytes to the spiral ligament type II fibrocytes and supralimbal fibrocytes to suprastrial fibrocytes in the lateral wall. From content of creatine kinase and position in the transport path, limbal light fibrocytes appeared analogous to spiral ligament type I fibrocytes. The additional finding that limbal fibrocytes showed unchanged or upregulated Na,K-ATPase immunoreactivity in aged gerbils with strial atrophy provided further evidence for an independent medial transport route and for the survival of inner hair cells in presbyacusis.

Evidence for glycoconjugate in nociceptive primary sensory neurons and its origin from the Golgi complex

Glycoconjugates with terminal galactose residues were localized in rat spinal cord and spinal ganglia using lectin-HRP conjugates of Griffonia simplicifolia and Glycine max agglutinins. Alternate staining of serial sections with HRP-labelled lectins and an antibody for substance P (SP) showed staining in identical primary sensory neurons with both methods. Similarly, lectin-reactive as well as SP-positive fibers were found in Rexed laminae I and II, Lissauers tract, the spinal nucleus and tract of the trigeminal nerve, the nucleus commissuralis and a small bundle of fibers just ventral to the central canal. Administration of capsaicin to neonatal rats produced a significant decrease in lectin-reactive fibers of the substantia gelatinosa, and in the number of lectin-reactive sensory neurons. The coexistence of SP with galactose-containing glycoconjugates in spinal ganglion neurons, as well as sensitivity of these cells to capsaicin, provided a basis for classifying the reactive neurons as nociceptive in type. Ligation of dorsal roots resulted in disappearance of lectin reactivity in the spinal cord and caused accumulation of lectin-positive material proximal to the ligature, indicating somatofugal transport of galactose-containing glycoconjugates. Colchicine injection caused an increase in SP reactivity in dorsal ganglion neurons but no change in lectin staining of galactoconjugate. At the ultrastructural level affinity for the lectin conjugates was confined to the Golgi cisternae and the plasmalemma of B-type sensory neurons in the dorsal ganglion. The axolemma of unmyelinated processes stained selectively in dorsal roots and the substantia gelatinosa of the spinal cord. These findings provide evidence for the presence in certain sensory cells of a characteristic galactosylconjugate which may prove to be of significance in nerve function.

Expression of α and β subunit isoforms of Na,K-ATPase in the mouse inner ear and changes with mutations at the Wv or Sld loci

Abstract Mice homozygous for mutations at the viable dominant spotting (Wv) and Steel-dickie (Sld) loci exhibit a similar phenotype which includes deafness. The auditory dysfunction derives from failure of the stria vascularis to develop normally and to generate a high positive endocochlear potential (EP). Because strial function is driven by Na,K-ATPase its expression was investigated in inner ears of W v W v and Sl d Sl d mice and their wild-type littermates by immunostaining with antisera against four of the enzymes subunit isoforms. Wild-type mice from two different genetic backgrounds showed an identical distribution of subunit isoforms among inner ear transport cells. Several epithelial cell types coexpressed the α1 and β1 subunits. Vestibular dark cells showed no reactivity for β1 but expressed abundant β2, whereas, strial marginal cells stained strongly for both β isoforms. The only qualitative difference between mutant and wild-type mice was the absence of β1 subunit in marginal cells of the mutants stria. However, it is unlikely that this difference accounts for failure of mutants to generate a high EP because the β1 subunit is not present in the stria vascularis of either rats or gerbils with normal EP values. Strong immunostaining for Na,K-ATPase in lateral wall fibrocytes of normal mice along with diminished immunoreactivity in the mutants supports the concept that these strategically located transport fibrocytes actively resorb K+ leaked across Reissners membrane into scala vestibuli or effluxed from hair cells and nerves into scala tympani. It is further speculated that the resorbed K+ normally is siphoned down its concentration gradient into the intrastrial space through gap junctions between fibrocytes and strial basal and intermediate cells where it is recycled back to endolymph via marginal cells. Thus, failure of mutants to generate a positive EP could be explained by the absence of intermediate cells which may form the final link in the conduit for moving K+ from perilymph to the intrastrial compartment.