Hair cell death in a hearing-deficient canary.

Cell death has been documented in bird auditory inner ear epithelia after induced damage. This cell death is quickly followed by an increase in supporting cell division and regeneration of the epithelium, thereby suggesting a possible relationship between these two processes. However, aspects of this relationship still need to be better understood. The Belgian Waterslager (BWS) canary is an ideal system in which to study cell death and subsequent cell division. In contrast to mixed breed (MB) canaries, cell division normally occurs in the auditory end organ of the BWS without any external manipulation. In addition, some of the cells in the auditory epithelium may be dying through an apoptotic-like process. In the present study two methods were used to quantify dying cells in the BWS and MB canary auditory epithelia: morphological criteria and TUNEL. Results confirm that some of the abnormal hair cells in the BWS auditory epithelium are apoptotic-like. The presence of both cell death and cell division indicates that these processes act concurrently in the adult end organ. Future studies are needed to determine if cell death is a stimulus for the observed cell division.

Proliferation of vertebrate inner ear supporting cells.

Using two S phase markers, we determined the cell-cycle behavior of inner ear supporting cells from two species, the chicken and the oscar. The results indicate that chicken utricular supporting cells divide once and do not return to the cell cycle for at least 7 days. In contrast, supporting cell progeny in the oscar saccule return to S phase after 5 days. While both the chicken utricle and oscar saccule show ongoing supporting cell proliferation, these data indicate that there may be a dedicated recycling population of supporting cells in the oscar saccule but not in the chicken utricle that is responsible for hair cell production. An expulsion of proliferative cell progeny in the chicken utricle after 7 days may be a driving force for proliferation, as well as an explanation for why hair cell numbers do not increase in the chicken utricle with age. This was not seen in the oscar saccule, possibly explaining how this end organ increases in size throughout the adult life of the animal. The absence of S phase cell expulsion, however, does not rule out the role of cell death in the oscar saccule.

Evidence for supporting cell proliferation and hair cell differentiation in the basilar papilla of adult Belgian Waterslager canaries (Serinus canarius).

We used the bromodeoxyuridine technique to study the proliferative activity in the basilar papilla of normal and Belgian Waterslager canaries with and without preceding sound trauma. Without sound trauma, there were, on average, six supporting cell divisions per day in the basilar papilla of Waterslager canaries. This rate of supporting cell proliferation corresponds well with estimates of the rate of hair cell differentiation derived from counts of immature-appearing hair cells obtained by using scanning electron microscopy of the Waterslager basilar papilla. Thus, supporting cell division appeared correlated with hair cell differentiation in Waterslager canaries. Bromodeoxyuridine labeling of cells in undamaged non-Waterslager canaries also indicated a very low rate of supporting cell division. In contrast with Waterslager canaries, this low rate of proliferation was not associated with a measurable rate of hair cell differentiation. In both normal and Waterslager canaries, exposure to traumatizing sound induced a dramatic increase in the rate of cell proliferation. These data show that a very low rate of supporting cell proliferation is normally present in birds, but it is not associated with a corresponding rate differentiation of hair cells. Only an increase above this low ambient rate of supporting cell proliferation, such as that following loss of hair cells, induces the differentiation of new hair cells in birds. The reason why Waterslager canaries do not completely compensate for their inherited hair cell deficit of 30% is not clear, when they can clearly respond to additional cochlear trauma from noise exposure with an increase in proliferation rate.

Cell proliferation and hair cell addition in the ear of the goldfish, Carassius auratus.

Cell proliferation and hair cell addition have not been studied in the ears of otophysan fish, a group of species who have specialized hearing capabilities. In this study we used the mitotic S-phase marker bromodeoxyuridine (BrdU) to identify proliferating cells in the ear of one otophysan species, Carassius auratus (the goldfish). Animals were sacrificed at 3 h or 5 days postinjection with BrdU and processed for immunocytochemistry. The results of the study show that cell proliferation occurs in all of the otic endorgans and results in the addition of new hair cells. BrdU-labeled cells were distributed throughout all epithelia, including the primary auditory endorgan (saccule), where hair cell phenotypes vary considerably along the rostrocaudal axis. This study lays the groundwork for our transmission electron microscopy study of proliferative cells in the goldfish ear (Presson et al., Hearing Research 100 (1996) 10-20) as well as future studies of hair cell development in this species. The ability to predict, based on epithelial location, the future phenotype of developing hair cells in the saccule of the goldfish make that endorgan a particularly powerful model system for the investigation of early hair cell differentiation.

Hair cell precursors are ultrastructurally indistinguishable from mature support cells in the ear of a postembryonic fish.

The ultrastructure of S-phase cells in the postembryonic fish ear was compared with that of mature support cells. S-phase cells were identified by injecting animals with [3H]thymidine and sacrificing 3 h later. Sensory epithelia (saccules, utricles, and canals) were processed for light-level autoradiography. Sections containing thymidine-labeled cells were re-embedded and re-examined using transmission electron microscopy. The results indicate that S-phase cells differ from mature support cells only in nuclear position and shape. Otherwise their cytoplasmic characteristics are indistinguishable. Both cell types, on the other hand, are readily distinguishable from hair cells. These data provide ultrastructural evidence for the ability of mature support cells to enter the cell cycle in postembryonic vertebrates.

[Continuous proliferation of supporting cells and indications for hair cell differentiation in the inner ear of adult song birds with genetic cochlear hearing loss]. / Kontinuierliche Proliferation von Stützzellen und Hinweise für Haarzelldifferenzierung im Hörorgan von adulten Singvögeln mit genetischer cochleärer Hörstörung.

Our previous investigations demonstrated that the Belgian Waterslagers (BWS) canary (Serinus canarius) was affected by an inherited sensorineural hearing loss. Compared to normal canaries of others strains, hair cell numbers in these birds were reduced on average by 30%. Since other birds are able to replace similar hair cell numbers after cochlear trauma, we investigated if BWS have the potential for supporting cell proliferation with subsequent hair cell differentiation or if they lack the repair mechanisms known to operate in other birds. In the present study the S-phase marker bromodeoxyuridine (BrdU) was used to demonstrate DNA synthesis and thus cell proliferation. We found on average six labelled nuclei per basilar papilla in BWS. This number of proliferating cells was in accordance with previous estimates of newly generated hair cells as based on the frequency of immature-appearing hair cells observed by scanning electron microscopy. We conclude that the division of supporting cells in BWS precedes the differentiation of hair cells. In contrast to BWS we found on average only one supporting cell division per day in normal canaries of other strains. However, this supporting cell proliferation in normal birds is probably not related to a loss of hair cells and does not lead to the differentiation of new hair cells. Our data indicate that differentiation of hair cells after supporting cell division occurs only if the rate of supporting cell proliferation is increased above the normal low level (probably by the loss of hair cells). Since BWS do not repair their basilar papilla despite a 30% hair cell loss (as compared to normal canaries) although they continuously produce new hair cells, we suggest that the regulation of the regeneration process is abnormal.

Proliferating hair cell precursors in the ear of a postembryonic fish are replaced after elimination by cytosine arabinoside.

Identified, proliferating S-phase cells in the postembryonic fish ear are known to be the precursors to new hair cells. It is not known, however, whether the ability to proliferate is restricted to a small population of cells. The ability of cells that are not normally in the cell cycle to enter S-phase was examined using the antimitotic drug cytosine arabinoside (ara-C). The normal population of S-phase cells in the saccule was destroyed by a single large dose of ara-C. Two weeks later, the presence of S-phase cells was evaluated using the S-phase marker bromodeoxyuridine. The results strikingly demonstrate that S-phase cells are replaced, since S-phase cells returned to the saccule in the same number as found in normal fish. The data are interpreted to suggest that a large number of nonsensory support cells are capable of entering the cell cycle and that some mechanism must regulate which of these are actually cycling at any given time.

Immunocytochemical reactivities of precursor cells and their progeny in the ear of a cichlid fish.

Cell types in the inner ear of the fish Astronotus ocellatus were examined for the immunocytochemical reactivity to 31 commercial antibodies. Nine showed positive reactivity: vimentin, S-100, caldesmon, calbindin, MAP-1, MAP-2, parvalbumin, neurofilament, and GAP-43. The cell types examined were: hair cells, support cells, hair cell precursors, eighth nerve neurons, and neuronal precursors. The pattern of reactivities among these cell types lead to the following conclusions. First, hair cells and eighth nerve neurons have a striking immunocytochemical similarity. Second, the precursor cells for hair cells and neurons did not share immunoreactivity with these mature progeny. Third, the only antibody to react with supporting cells also reacted with the proliferating precursors that give rise to new hair cells and supporting cells. Taken with other available data, these finding suggest that in the oscar ear, hair cell precursors and supporting cells are closely related, if not the same cell type.

Modes of neuronal arbor enlargement in the ear of a postembryonic fish, Astronotus ocellatus.

New hair cells are added during postembryonic life in several species of fishes and birds. The production of new hair cells appears to require enlargement of eighth nerve arbors during growth since, at least in fish, eighth nerve neurons are added more slowly than hair cells or not at all. This situation provides an intriguing opportunity to study the mechanisms of growth of the neuronal arbors. In this paper, we report the results of studies on the postembryonic growth of eighth nerve dendritic arbors in the saccular epithelium of the cichlid fish Astronotus ocellatus. Arbor sizes and shapes were compared in small and large fish using the axonal tracer cobaltous lysine. Our data suggest that postembryonic eighth nerve arbors enlarge in 2 ways. First, arbors add new terminal endings to their distal ends. Second, whole new branches appear to be added at locations up to hundreds of micrometers proximal to the terminal endings. These 2 modes of growth suggest that more than one mechanism may be operative in controlling arbor enlargement.

Central-peripheral and rostral-caudal organization of the innervation of the saccule in a cichlid fish.

Saccular eighth nerve arbors were examined in the cichlid fish Astronotus ocellatus to determine if their morphology varies with saccular location. The saccule was divided into four regions: rostral-central, rostral-peripheral, caudal-central, caudal-peripheral. Arbors were filled with cobaltous-lysine. Axon diameter, maximum arbor width, and number of terminal points were taken as quantitative measures. Differences in these measures among the four different saccular regions were evaluated using an analysis of variance. The results indicate two types of organization: central-peripheral and rostral-caudal. The central-peripheral differences involve all three quantitative measures. The central saccule is innervated by arbors with larger axon diameters, larger arbor widths, and more terminal points than the peripheral saccule. The rostral-caudal organization involves only two measures. The rostral saccule is innervated by arbors having larger axon diameters and smaller arbor widths than the caudal saccule. In the oscar, we know of no other parameters that are organized along the rostral-caudal dimension. These findings of a spatial organization of innervation suggest to us that the oscar saccule is not homogeneous in function.

Sensory hair cells of a fish ear: evidence of multiple types based on ototoxicity sensitivity.

Sensory hair cells from the striolar region (striolar hair cells) of the utricle and the lagena of the ear of a teleost fish Astronotus ocellatus (Cuvier) ear are sensitive to gentamicin sulphate, an ototoxic drug. In contrast, sensory hair cells from outside the striolar region (extra-striolar hair cells) are not sensitive to gentamicin. These data, combined with results from studies showing different ultrastructural features and different immunoreactivity to a calcium binding protein, S-100, lead to the suggestion that there are distinguishable types of hair cells in these endorgans. These results add to the increasing evidence that classifying the sensory hair cells of fish ears only as the traditional 'vestibular type II' may be inadequate for properly understanding structure and function of the fish ear.

S-100 immunoreactivity identifies a subset of hair cells in the utricle and saccule of a fish.

Certain hair cells of fish exhibit strong immunoreactivity to an S-100 antibody. By their spatial locations in the utricle and saccule, these hair cells appear to possess a relatively short kinocilium and a roughly ovoid cell shape. In the utricle, these cells are predominantly located in the striola. In the saccule, these cells are found within the central area of the epithelium. In both of these epithelia the strongly immunoreactive hair cells coincide with the locations of hair cells possessing F1 ciliary bundles.

A ganglionic source of new eighth nerve neurons in a post-embryonic fish.

In the post-embryonic fish Astronotus ocellatus (the oscar) sensory hair cells and eighth nerve neurons are added to the peripheral statoacoustic system all during adulthood. Here we use 3H-thymidine to label S-phase precursors to new neurons in the statoacoustic ganglion. The neuronal precursor cells have small nuclei with little surrounding cytoplasm. They are surrounded by cells that appear to be part of a developmental sequence ending with mature eighth nerve neurons. Taken with our previous studies, these results demonstrate that hair cell and neuronal precursors in post-embryonic oscars are spatially segregated and morphologically distinct from one another.

Possible precursors to new hair cells, support cells, and Schwann cells in the ear of a post-embryonic fish.

The sources of new hair cells, support cells, and Schwann cells were identified in the statoacoustic end organs of normal post-embryonic fish (Astronotus ocellatus). S-phase cells, defined as cells that take up 3H-thymidine in preparation for mitosis, and their progeny were visualized using autoradiography. Two types of S-phase cells were found: 'embryonic-like neuroepithelial' (NE) cells and 'basally located S-phase' (BLS) cell. The NE cells had elongated nuclei and processes extending basally and apically. Thirty minutes after the thymidine injection labeled NE cell nuclei were found between hair cell nuclei and support cell nuclei. After two and four hours survival some labeled NE nuclei were closer to the lumen, where they divided. One labeled support cell was seen after four hours survival and one labeled hair cell after nine hours survival. Significant numbers of labeled support and hair cells were not seen until 24 h survival. These results led us to identify NE cells as the immediate source of new hair cells and support cells. The BLS cells had small nuclei with little surrounding cytoplasm. Shortly after the thymidine injection BLS cells were found between the hair cell nuclei and the basement membrane, and they underwent mitosis in this position. The BLS cells resemble intra-epithelial Schwann cells, except that they are not curved around axonal profiles. We suggest that the BLS cells are Schwann cell precursors.

Development of the optic tract in the cichlid fish Haplochromis burtoni.

In the teleost fish, Haplochromis burtoni, the optic tract is composed of 3 distinct components: the marginal tract, which projects to the optic tectum and is by far the largest, and the axial and medial tracts which project to diencephalic targets. In this paper we report on the normal development of these pathways in larval H. burtoni, an African cichlid fish. The earliest optic tract fibers are found in what will become the marginal optic tract. These fibers hug the wall of the diencephalon in a cohesive bundle. The first fibers in the axial tract location appear on day 5, increasing in number between days 6 and 18. Like marginal tract fibers, axial tract fibers form a cohesive bundle. It is not clear from these experiments whether the first axial tract fibers actually arrive at this location at day 5, or whether they are fibers arriving earlier that were physically displaced from the marginal tract at day 5. Medial tract fibers are not evident until day 6 of development and the number of medial tract fibers also increases as the animal gets older. Unlike fibers in the other two pathways, medial tract fibers do not travel together in a bundle. Rather, each one follows an independent trajectory to its target site. Comparison of this larval development with the adult optic tract organization which we have studied earlier suggests constraints on the mechanisms of axon guidance.