Synchronization of hair cell regeneration in the chick cochlea following noise damage

0 Department of Anatomy & Neurobiology, Boston University School of Medicine , 80 East Concord Street, Boston, MA 02118 , USA Synchronization of hair cell regeneration in the chick cochlea following JENNIFER S. STONE and DOUGLAS A. COTANCHE* Pure-tone overstimulation for prolonged time leads to hair cell death in frequency-specific regions of the cochlear epithelium. Unlike mammals, birds replace missing hair cells by stimulating mitosis in an uncharacterized precursor cell. Regenerated hair cells, initially identifiable by their immature stereodlia and small surface areas, differentiate into mature cells in a manner which parallels embryonic development. In the current study, we examined whether hair cell regeneration is initiated during noise exposure or after the end of acoustic trauma. We exposed 7- to 15-day-old chicks to a 1500 Hz pure tone at 120 dB SPL (re 20 /iPa) for 4, 12, and 24 hours and examined the recovering cochlear epithelium with scanning electron microscopy to determine when regenerated hair cells were first identifiable. The earliest evidence of new hair cells appeared roughly - Author for correspondence The sensory epithelium of the cochlea consists of two primary cell populations: hair cells and supporting cells. Hair cells, the auditory receptors, have an apical bundle of stereocilia that is arranged in a staircase configuration and anchored to the overlying tectorial membrane. The coupling of hair cell stereocilia and the tectorial membrane mediates the transduction of mechanical disturbances within the fluid of the scala media into neural signals (Lowenstein and Wersall, 1959; Hudspeth and Corey, 1977). Hair cells in different regions of the cochlear epithelium are responsive to specific frequencies of sound, and this tonotopy is reflected in the systematic gradation in hair cell morphology along the length of the epithelium (Lim, 1980; Tilney and Saunders, 1983). Each hair cell is surrounded by several supporting cells, which have apical microvilli and secrete a portion of the tectorial membrane during development (Cohen and Fermin, 1985; Sheil and Cotanche, 1990). Hair cells are traumatized as a result of acoustic overstimulation and exposure to ototoxic drugs. Following noise exposure, some hair cells sustain only a 96 hours after the onset of 4-, 12-, and 24-hour exposures. Our previous studies initially identified new hair cells 96 hours after the start of a 48-hour exposure. Therefore, hair cell regeneration follows a similar time course relative to the onset of noise exposure, regardless of the ultimate duration of exposure. Since we estimate that hair cells take at least 48 hours after their genesis to form immature stereocilia, the signal which induces hair cell precursors to re-enter the cell cycle and to divide probably has its initial effects very early during the exposure period. (A previous report of these data was given at the 1991 American Society for Cell Biology conference.) mild degree of structural damage and display contraction of their apical surfaces and/or splaying of the stereociliary bundle. Other hair cells are severely damaged and are expelled from the epithelium shortly after exposure to noise (Cotanche et al., 1987; Cotanche, 1987a; Cotanche and Dopyera, 1990). Balloon-like structures protruding from the cochlear epithelium into the scala media are identifiable as hair cell remnants because they have stereocilia (Cotanche et al., 1987). The tectorial membrane dissociates from hair cells in the damaged region and retracts toward the superior edge (Cotanche, 1987b; Cotanche et al., 1991). In mammals, lost hair cells are not replaced, and permanent hearing deficits ensue (Engstrom et al., 1966; Bohne, 1976; Hawkins and Johnsson, 1976; Bohne and Rabbit, 1983). However, birds and other submammalian vertebrates reconstitute the appropriate number and morphology of hair cells in the damaged region, and threshold shifts recover (Cotanche, 1987a; Cruz et al., 1987; Henry et al., 1988; McFadden and Saunders, 1989; Duckert and Rubel, 1990; Tucci and Rubel, 1990). Tritiated thymidine studies which compared normal and regenerating avian cochleae demonstrated that the precursors to new hair cells undergo /. 5. Stone and D. A. Cotanche DNA synthesis and divide prior to differentiating into recognizable hair cells, but post-embryonic mitotic activity is not routine (Corvvin and Cotanche, 1988; Ryals and Rubel, 1988; Girod et al., 1989; Lippe et al., 1991). Thus, with the appropriate stimulus, cell division is triggered in the mature, normally quiescent cochlear epithelium. The precursor to new hair cells, which has not been identified, probably exists in the epithelium or in its vicinity in a growth-arrested (Go) state. In order to produce two daughter cells, this precursor must reenter the cell cycle at the gap 1 (Gi) phase and proceed through the DNA synthesis (S) phase, the gap 2 (G2) phase, and the mitotic (M) phase. Unlike some regenerating organs, such as the epidermis or the gastrointestinal tract, there does not appear to be a local population of stem cells poised to divide in the cochlea. Therefore, it is likely that a relatively differentiated cell leaves the growth-arrested state, re-enters the cell cycle, and generates new hair cells. These cells then develop into mature hair cells in a precise, stepwise manner that is morphologically and temporally similar to that for embryos (Cotanche, 1987a; Corwin and Warchol, 1991; Cotanche et al., 1991). The specific mechanisms that regulate hair cell regeneration have not been characterized. However, the duration and intensity of noise during exposure clearly determine the extent of hair cell damage and recovery; longer exposure periods and higher intensities induce larger areas of damage and greater numbers of regenerated hair cells (Rubel and Ryals, 1982; Cotanche et al., 1987; Cotanche and Dopyera, 1990; Cotanche et al., 1991). Until recently, it was generally reasoned that hair cell regeneration cannot occur while acoustic trauma or ototoxic drug damage is in progress. However, results from three recent studies provide convincing, although indirect, evidence that precursor cells divide during the insult. Immature bundles of stereocilia were evident 24 hours after a 5-day treatment with an ototoxic drug, gentamicin (Duckert and Rubel, 1990). Taking into account our knowledge of the embryonic development of hair cells, this observation suggests that hair cell regeneration begins during gentamicin treatment. In the embryo, the first terminal mitoses occur in many regions of the cochlea by embryonic day 4 (Katayama and Corwin, 1989). Hair cells arefirstidentifiable at embryonic day 6 in the distal end, since they have formed an immature bundle of stereocilia (Cotanche and Sulik, 1984). Thus, hair cells require approximately 48 hours after their genesis to differentiate cell-specific profiles. It follows that the cell division necessary to yield new hair cells must be (...truncated)