Immunocytochemical and morphological evidence for intracellular self-repair as an important contributor to mammalian hair cell recovery.
(1/1112)
Although recent studies have provided evidence for hair cell regeneration in mammalian inner ears, the mechanism underlying this regenerative process is still under debate. Here we report immunocytochemical, histological, electron microscopic, and autoradiographic evidence that, in cultured postnatal rat utricles, a substantial number of hair cells can survive gentamicin insult even their stereocilia are lost. These partially damaged hair cells can survive for a prolonged time and regrow the stereocilia. Although the number of stereocilia-bearing hair cells increases over time after gentamicin insult, hair cell and supporting cell numbers remain essentially unchanged. Tritiated thymidine autoradiography and bromodeoxyuridine immunocytochemistry of the cultures demonstrate that cell proliferation in the sensory epithelium is very limited and is far below the number of recovered hair cells. Furthermore, terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling analysis indicates that gentamicin-induced apoptosis in the sensory epithelium occurs mainly during a 2 d treatment period, and additional cell death is minimal 2-11 d after treatment. Considered together, intracellular repair of partially damaged hair cells can be an important contributor to spontaneous hair cell recovery in mammalian inner ears. (+info)
Synapses involving auditory nerve fibers in primate cochlea.
(2/1112)
The anatomical mechanisms for processing auditory signals are extremely complex and incompletely understood, despite major advances already made with the use of electron microscopy. A major enigma, for example, is the presence in the mammalian cochlea of a double hair cell receptor system. A renewed attempt to discover evidence of synaptic coupling between the two systems in the primate cochlea, postulated from physiological studies, has failed. However, in the outer spiral bundle the narrow and rigid clefts seen between pairs of presumptive afferent fibers suggest the possibility of dendro-dendritic interaction confined to the outer hair cell system. The clustering of afferent processes within folds of supporting cells subjacent to outer hair cells is in contrast to the lack of such close associations in the inner hair cell region. The difference reinforces the suggestion of functional interaction of some sort between the outer hair cell afferent nerve processes. (+info)
Gene disruption of p27(Kip1) allows cell proliferation in the postnatal and adult organ of corti.
(3/1112)
Hearing loss is most often the result of hair-cell degeneration due to genetic abnormalities or ototoxic and traumatic insults. In the postembryonic and adult mammalian auditory sensory epithelium, the organ of Corti, no hair-cell regeneration has ever been observed. However, nonmammalian hair-cell epithelia are capable of regenerating sensory hair cells as a consequence of nonsensory supporting-cell proliferation. The supporting cells of the organ of Corti are highly specialized, terminally differentiated cell types that apparently are incapable of proliferation. At the molecular level terminally differentiated cells have been shown to express high levels of cell-cycle inhibitors, in particular, cyclin-dependent kinase inhibitors [Parker, S. B., et al. (1995) Science 267, 1024-1027], which are thought to be responsible for preventing these cells from reentering the cell cycle. Here we report that the cyclin-dependent kinase inhibitor p27(Kip1) is selectively expressed in the supporting-cell population of the organ of Corti. Effects of p27(Kip1)-gene disruption include ongoing cell proliferation in postnatal and adult mouse organ of Corti at time points well after mitosis normally has ceased during embryonic development. This suggests that release from p27(Kip1)-induced cell-cycle arrest is sufficient to allow supporting-cell proliferation to occur. This finding may provide an important pathway for inducing hair-cell regeneration in the mammalian hearing organ. (+info)
2E4 (kaptin): a novel actin-associated protein from human blood platelets found in lamellipodia and the tips of the stereocilia of the inner ear.
(4/1112)
Platelet activation, crucial for hemostasis, requires actin polymerization, yet the molecular mechanisms by which localized actin polymerization is mediated are not clear. Here we report the characterization of a novel actin-binding protein, 2E4, originally isolated from human blood platelets and likely to be involved in the actin rearrangements occurring during activation. 2E4 binds to filamentous (F)-actin by F-actin affinity chromatography and is eluted from F-actin affinity columns and extracted from cells with ATP. Its presence at the leading edge of platelets spread on glass and in the lamellipodia of motile fibroblasts suggests a role in actin dynamics. Using localization to obtain clues about function, we stained the sensory epithelium of the embryonic inner ear to determine whether 2E4 is at the barbed end of actin filaments during their elongation. Indeed, 2E4 was present at the tips of the elongating stereocilium. 2E4 is novel by DNA sequence and has no identifiable structural motifs. Its unusual amino acid sequence, its ATP-sensitive actin association and its location at sites of actin polymerization in cells suggest 2E4 plays a unique role in the actin rearrangements that accompany platelet activation and stereocilia formation. (+info)
Electrical response properties of avian lagena type II hair cells: a model system for vestibular filtering.
(5/1112)
Data presented represent the first electrical recordings from avian lagena type II hair cells. The perforated-patch variant of the whole cell recording technique was used to investigate how the macroscopic currents shaped the voltage response of the hair cells. Voltage-clamp data separated cells into two broad classes on the basis of differences in activation rates, rates and degree of inactivation, and pharmacological sensitivity. Current-clamp recordings revealed low-quality membrane voltage oscillations (Qc < 1) during pulse current injections. Oscillation frequency correlated with activation rate of the macroscopic currents. The quality of membrane oscillations (Qc) varied linearly with frequency for cells with little inactivation. For cells with rapid inactivation, no relationship was found between Qc and frequency. Rapid inactivation may serve to extend the bandwidth of vestibular hair cells. The frequency measured from voltage responses to pulsed currents may reflect the corner frequency of the cell. The filtering properties of avian lagena hair cells are like those found in all other vestibular end organs, suggesting that the electrical membrane properties of these cells are not responsible for specializing them to a particular stimulus modality. (+info)
Functional expression of exogenous proteins in mammalian sensory hair cells infected with adenoviral vectors.
(6/1112)
To understand the function of specific proteins in sensory hair cells, it is necessary to add or inactivate those proteins in a system where their physiological effects can be studied. Unfortunately, the usefulness of heterologous expression systems for the study of many hair cell proteins is limited by the inherent difficulty of reconstituting the hair cell's exquisite cytoarchitecture. Expression of exogenous proteins within hair cells themselves may provide an alternative approach. Because recombinant viruses were efficient vectors for gene delivery in other systems, we screened three viral vectors for their ability to express exogenous genes in hair cells of organotypic cultures from mouse auditory and vestibular organs. We observed no expression of the genes for beta-galactosidase or green fluorescent protein (GFP) with either herpes simplex virus or adeno-associated virus. On the other hand, we found robust expression of GFP in hair cells exposed to a recombinant, replication-deficient adenovirus that carried the gene for GFP driven by a cytomegalovirus promoter. Titers of 4 x 10(7) pfu/ml were sufficient for expression in 50% of the approximately 1,000 hair cells in the utricular epithelium; < 1% of the nonhair cells in the epithelium were GFP positive. Expression of GFP was evident as early as 12 h postinfection, was maximal at 4 days, and continued for at least 10 days. Over the first 36 h there was no evidence of toxicity. We recorded normal voltage-dependent and transduction currents from infected cells identified by GFP fluorescence. At longer times hair bundle integrity was compromised despite a cell body that appeared healthy. To assess the ability of adenovirus-mediated gene transfer to alter hair cell function we introduced the gene for the ion channel Kir2.1. We used an adenovirus vector encoding Kir2.1 fused to GFP under the control of an ecdysone promoter. Unlike the diffuse distribution within the cell body we observed with GFP, the ion channel-GFP fusion showed a pattern of fluorescence that was restricted to the cell membrane and a few extranuclear punctate regions. Patch-clamp recordings confirmed the expression of an inward rectifier with a conductance of 43 nS, over an order of magnitude larger than the endogenous inward rectifier. The zero-current potential in infected cells was shifted by -17 mV. These results demonstrate an efficient method for gene transfer into both vestibular and auditory hair cells in culture, which can be used to study the effects of gene products on hair cell function. (+info)
The supporting-cell antigen: a receptor-like protein tyrosine phosphatase expressed in the sensory epithelia of the avian inner ear.
(7/1112)
After noise- or drug-induced hair-cell loss, the sensory epithelia of the avian inner ear can regenerate new hair cells. Few molecular markers are available for the supporting-cell precursors of the hair cells that regenerate, and little is known about the signaling mechanisms underlying this regenerative response. Hybridoma methodology was used to obtain a monoclonal antibody (mAb) that stains the apical surface of supporting cells in the sensory epithelia of the inner ear. The mAb recognizes the supporting-cell antigen (SCA), a protein that is also found on the apical surfaces of retinal Muller cells, renal tubule cells, and intestinal brush border cells. Expression screening and molecular cloning reveal that the SCA is a novel receptor-like protein tyrosine phosphatase (RPTP), sharing similarity with human density-enhanced phosphatase, an RPTP thought to have a role in the density-dependent arrest of cell growth. In response to hair-cell damage induced by noise in vivo or hair-cell loss caused by ototoxic drug treatment in vitro, some supporting cells show a dramatic decrease in SCA expression levels on their apical surface. This decrease occurs before supporting cells are known to first enter S-phase after trauma, indicating that it may be a primary rather than a secondary response to injury. These results indicate that the SCA is a signaling molecule that may influence the potential of nonsensory supporting cells to either proliferate or differentiate into hair cells. (+info)
The electrical properties of auditory hair cells in the frog amphibian papilla.
(8/1112)
The amphibian papilla (AP) is the principal auditory organ of the frog. Anatomical and neurophysiological evidence suggests that this hearing organ utilizes both mechanical and electrical (hair cell-based) frequency tuning mechanisms, yet relatively little is known about the electrophysiology of AP hair cells. Using the whole-cell patch-clamp technique, we have investigated the electrical properties and ionic currents of isolated hair cells along the rostrocaudal axis of the AP. Electrical resonances were observed in the voltage response of hair cells harvested from the rostral and medial, but not caudal, regions of the AP. Two ionic currents, ICa and IK(Ca), were observed in every hair cell; however, their amplitudes varied substantially along the epithelium. Only rostral hair cells exhibited an inactivating potassium current (IA), whereas an inwardly rectifying potassium current (IK1) was identified only in caudal AP hair cells. Electrically tuned hair cells exhibited resonant frequencies from 50 to 375 Hz, which correlated well with hair cell position and the tonotopic organization of the papilla. Variations in the kinetics of the outward current contribute substantially to the determination of resonant frequency. ICa and IK(Ca) amplitudes increased with resonant frequency, reducing the membrane time constant with increasing resonant frequency. We conclude that a tonotopically organized hair cell substrate exists to support electrical tuning in the rostromedial region of the frog amphibian papilla and that the cellular mechanisms for frequency determination are very similar to those reported for another electrically tuned auditory organ, the turtle basilar papilla. (+info)