Peripheral innervation patterns of vestibular nerve afferents in the bullfrog utriculus.

Vestibular nerve afferents innervating the bullfrog utriculus differ in their response dynamics and sensitivity to natural stimulation. They also supply hair cells that differ markedly in hair bundle morphology. To examine the peripheral innervation patterns of individual utricular afferents more closely, afferent fibers were labeled by the extracellular injection of horseradish peroxidase (HRP) into the vestibular nerve after sectioning the vestibular nerve medial to Scarpa's ganglion to allow the degeneration of sympathetic and efferent fibers. The peripheral arborizations of individual afferents were then correlated with the diameters of their parent axons, the regions of the macula they innervate, and the number and type of hair cells they supply. The utriculus is divided by the striola, a narrow zone of distinctive morphology, into medial and lateral parts. Utricular afferents were classified as striolar or extrastriolar according to the epithelial entrance of their parent axons and the location of their terminal fields. In general, striolar afferents had thicker parent axons, fewer subepithelial bifurcations, larger terminal fields, and more synaptic endings than afferents in extrastriolar regions. Afferents in a juxtastriolar zone, immediately adjacent to the medial striola, had innervation patterns transitional between those in the striola and more peripheral parts of the medial extrastriola. Most afferents innervated only a single macular zone. The terminal fields of striolar afferents, with the notable exception of a few afferents with thin parent axons, were generally confined to one side of the striola. Hair cells in the bullfrog utriculus have previously been classified into four types based on hair bundle morphology (Lewis and Li: Brain Res. 83:35-50, 1975). Afferents in the extrastriolar and juxtastriolar zones largely or exclusively innervated Type B hair cells, the predominant hair cell type in the utricular macula. Striolar afferents supplied a mixture of four hair cell types, but largely contacted Type B and Type C hair cells, particularly on the outer rows of the medial striola. Afferents supplying more central striolar regions innervated fewer Type B and large numbers of Type E and Type F hair cells. Striolar afferents with thin parent axons largely supplied Type E hair cells with bulbed kinocilia in the innermost striolar rows.

Calbindin and parvalbumin are early markers of non-mitotically regenerating hair cells in the bullfrog vestibular otolith organs.

Earlier studies have demonstrated hair cell regeneration in the absence of cell proliferation, and suggested that supporting cells could phenotypically convert into hair cells following hair cell loss. Because calcium-binding proteins are involved in gene up-regulation, cell growth, and cell differentiation, we wished to determine if these proteins were up-regulated in scar formations and regenerating hair cells following gentamicin treatment. Calbindin and parvalbumin immunolabeling was examined in control or gentamicin-treated (GT) bullfrog saccular and utricular explants cultured for 3 days in amphibian culture medium or amphibian culture medium supplemented with aphidicolin, a blocker of nuclear DNA replication in eukaryotic cells. In control cultures, calbindin and parvalbumin immunolabeled the hair bundles and, less intensely, the cell bodies of mature hair cells. In GT or mitotically-blocked GT (MBGT) cultures, calbindin and parvalbumin immunolabeling was also seen in the hair bundles, cuticular plates, and cell bodies of hair cells with immature hair bundles. Thus, these antigens were useful markers for both normal and regenerating hair cells. Supporting cell immunolabeling was not seen in control cultures nor in the majority of supporting cells in GT cultures. In MBGT cultures, calbindin and parvalbumin immunolabeling was up-regulated in the cytosol of single supporting cells participating in scar formations and in supporting cells with hair cell-like characteristics. These data provide further evidence that non-mitotic hair cell regeneration in cultures can be accomplished by the conversion of supporting cells into hair cells.

Regional differences in lectin binding patterns of vestibular hair cells.

Surface glycoconjugates of hair cells and supporting cells in the vestibular endorgans of the bullfrog were identified using biotinylated lectins with different carbohydrate specificities. Lectin binding in hair cells was consistent with the presence of glucose and mannose (CON A), galactose (RCA-I), N-acetylglucosamine (WGA), N-acetylgalactosamine (VVA), but not fucose (UEA-I) residues. Hair cells in the bullfrog sacculus, unlike those in the utriculus and semicircular canals, did not strain for N-acetylglucosamine (WGA) or N-acetylgalactosamine (VVA). By contrast, WGA and, to a lesser extent, VVA, differentially stained utricular and semicircular canal hair cells, labeling hair cells located in peripheral, but not central, regions. In mammals, WGA uniformly labeled Type I hair cells while labeling, as in the bullfrog, Type II hair cells only in peripheral regions. These regional variations were retained after enzymatic digestion. We conclude that vestibular hair cells differ in their surface glycoconjugates and that differences in lectin binding patterns can be used to identify hair cell types and to infer the epithelial origin of isolated vestibular hair cells.

Intracellular distributions and putative functions of calcium-binding proteins in the bullfrog vestibular otolith organs.

Hair cells in the bullfrog vestibular otolith organs were immunolabeled by monoclonal and polyclonal antisera against calbindin (CaB), calmodulin (CaM), calretinin (CaR), and parvalbumin (PA). S-100, previously shown to immunolabel striolar hair cells in fish vestibular organs, only weakly immunolabeled hair cells in the bullfrog vestibular otolith organs. Immunolabeling was not detected in supporting cells. With the exception of CaR, myelinated axons and unmyelinated nerve terminals were immunolabeled by all of the above antisera. Immunolabeling was seen in all saccular hair cells, although hair cells at the macular margins were immunolabeled more intensely for CaB, CaM, and PA than more centrally located hair cells. As the macula margins are known to be a growth zone, this labeling pattern suggests that marginal hair cells up-regulate their calcium-binding proteins during hair cell development. In the utriculus, immunolabeling for CaM and PA was generally restricted to striolar hair cells. CaR immunolabeling was restricted to the stereociliary array. Immunolabeling for other calcium-binding proteins was generally seen in both the cell body and hair bundles of hair cells, although this labeling was often localized to the stereociliary array and the apical portion of the cell body. CaM and PA immunolabeling in the stereociliary array in saccular and utricular striolar cells suggests a functional role for these proteins in mechanoelectric transduction and adaptation.

Hair cell regeneration in the bullfrog vestibular otolith organs following aminoglycoside toxicity.

Adult bullfrog were given single intraotic injections of the aminoglycoside antibiotic gentamicin sulfate and sacrificed at postinjection times ranging from 0.5 to 9 days. The saccular and utricular maculae of normal and injected animals were examined in wholemount and cross-section. Intraotic 200 microM gentamicin concentrations resulted in the uniform destruction of the hair bundles and, at later times, the cell bodies of saccular hair cells. In the utriculus, striolar hair cells were selectively damaged while extrastriolar hair cells were relatively unaffected. Regenerating hair cells, identified in sectioned material by their small cell bodies and short, well-formed hair bundles, were seen in the saccular and utricular maculae as early as 24-48 h postinjection. Immature versions of mature hair cell types in both otolith organs were recognized by the presence or absence of a bulbed kinocilia and the relative lengths of their kinocilia and longest stereocilia. Utricular hair cell types with kinocilia longer than their longest stereocilia were observed at earlier than hair cell types with shorter kinocilia. In the sacculus, the hair bundles of gentamicin-treated animals, even at 9 days postinjection, were significantly smaller than those of normal animals. The hair bundles of utricular hair cells, on the other hand, reached full maturity within the same time period.