Survival of Bundleless Hair Cells and Subsequent Bundle Replacement in the Bullfrog’s Saccule Jonathan E. Gale,1* Jason R. Meyers,1 Ammasi Periasamy,2 Jeffrey T. Corwin1 1 Department of Otolaryngology, HNS and Department of Neuroscience, University of Virginia School of Medicine, Charlottesville, Virginia 22908 2 Department of Biology, Keck Center for Cellular Imaging, University of Virginia, Charlottesville, Virginia 22906 Received 29 May 2001; accepted 12 July 2001 ABSTRACT: Our senses of hearing and balance lose their hair bundle and survive as bundleless cells for depend upon hair cells, the sensory receptors of the at least 1 week. Time-lapse and electron microscopy inner ear. Millions of people suffer from hearing and revealed stages in the separation of the bundle from the balance deficits caused by damage to hair cells as a cell body. Scanning electron microscopy (SEM) of cul- result of exposure to noise, aminoglycoside antibiotics, tures fixed 2, 4, and 7 days after antibiotic treatment and antitumor drugs. In some species such damage can showed that numerous new hair bundles were produced be reversed through the production of new cells. This between 4 and 7 days of culture. Further examination proliferative response is limited in mammals but it has revealed hair cells with small repaired hair bundles been hypothesized that damaged hair cells might survive alongside damaged remnants of larger surviving bun- and undergo intracellular repair. We examined the fate dles. The results indicate that sensory hair cells can of bullfrog saccular hair cells after exposure to a low undergo intracellular self-repair in the absence of mito- dose of the aminoglycoside antibiotic gentamicin to de- sis, offering new possibilities for functional hair cell termine whether hair cells could survive such treatment recovery and an explanation for non-proliferative and subsequently be repaired. In organ cultures of the recovery. © 2002 John Wiley & Sons, Inc. J Neurobiol 50: 81–92, bullfrog saccule a combination of time-lapse video mi- 2002; DOI 10.1002/neu.10002 croscopy, two-photon microscopy, electron microscopy, Keywords: vestibular; repair; regeneration; deafness; and immunocytochemistry showed that hair cells can hearing; ototoxicity INTRODUCTION activity (Hudspeth, 1989; Ashmore and Gale, 2000). Loss of hair cells is the major cause of noncongenital hearing and balance deficits (Nadol, 1993), and dam- Hair cells are the sensory receptor cells of the inner age to the transduction apparatus, the stereocilia bun- ear that transduce mechanical stimuli into electrical dle on the surface of the hair cell, is sufficient to cause hearing deficits (Liberman and Dodds, 1984). * Present address: Department of Physiology, University Col- Research in rodents has indicated that hair cells in lege London, Gower Street, London, WC1E 6BT, U.K. mammalian ears are normally produced only during Correspondence to: J. Gale ([email protected]) or J. T. Corwin embryonic and neonatal development (Ruben, 1967), ([email protected]). Contract grant sponsor: National Institute on Deafness and so that nearly all forms of damage to mammalian hair Other Communication Disorders; contract grant number: RO1- cells have been considered irreversible, consistent 0200. with the clinical permanence of hearing deficits (Lam- Contract grant sponsor: National Organization for Hearing Re- search. bert, 1994). That is not the case, however, for non- Contract grant sponsor: Lions of Virginia Hearing Research mammalian vertebrates. In fish and amphibians, thou- Center and Foundation. Contract grant sponsor: Wellcome Trust Prize Travelling Fel- sands of hair cells are added to the ear throughout lowship (J.E.G.). normal life, and lost hair cells are replaced (Corwin, © 2002 John Wiley & Sons, Inc. 1981, 1983, 1985; Popper and Hoxter, 1984; Diaz et 81 82 Gale et al. al., 1995). In birds, damage to the ear can be repaired placed in a 30 ␮L collagen droplet and cultured in Wolf and in less than 2 weeks via trauma-evoked production of Quimby medium (Gibco, USA) with added sodium pyru- new hair cells (reviewed in Corwin and Oberholtzer, vate (100 ␮M), 1X nonessential MEM amino acid solution 1997). (Gibco), and the antibiotic ciprofloxacin (0.01%) based on Recently it was discovered that vestibular epithelia the method of Baird et al. (1996). Test saccules were placed in culture medium supplemented with 300 ␮M gentamicin in mammalian ears exhibit some recovery after anti- for 16–18 h and then the media in treated and control biotic damage and that the machinery for regenerative cultures were replaced with fresh control medium. Medium proliferation could operate at a low level in mamma- was changed every 48–72 h. Saccules for electron micro- lian ears (Forge et al., 1993; Warchol et al., 1993). scopic analysis, time-lapse, and two-photon time-series ex- However, the incidence of cell proliferation in vitro periments were cultured in the presence of the DNA poly- was lower than might have been expected from the merase inhibitor aphidicolin at 25 ␮M (Harris and incidence of immature hair bundles that appeared in Hartenstein, 1991). vivo after damage, suggesting that mechanisms inde- pendent of regenerative proliferation were involved. Time-Lapse and Two-Photon Imaging Recent work that utilized low doses of aminoglyco- side antibiotics has suggested that the replacement of For time-lapse recordings saccules were cultured as above hair cells can occur in the absence of mitosis in except that they were oriented with their hair bundles down non-mammalian (Adler and Raphael, 1996; Baird et in collagen droplets and placed in Rose chambers on an al., 1996; Roberson et al., 1996) and mammalian inverted microscope at 24–26°C. The microscope was un- systems (Li and Forge, 1997; Zheng et al., 1999). der software control (Metamorph; Universal Imaging Inc., Thus, it has been suggested that replacement hair cells USA) allowing up to 12 z-axis focal planes to be recorded (typically only three were) in concurrent time lapse over a could arise via conversion of cells from a supporting 4–10 day period. Images were acquired every 3 to 5 min cell phenotype into a hair cell phenotype. An alterna- typically covering a z depth of between 30 and 45 ␮m. tive hypothesis is that damaged hair cells might sur- Saccules used for two-photon fluorescence microscopy vive and undergo cellular repair (Corwin et al., 1996). were incubated for 5 min in 30 ␮M FM1-43 (Molecular There is some evidence consistent with such self- Probes, USA), which loads into bullfrog hair cells selec- repair in damaged mammalian epithelia (Sobkowicz tively (Gale et al., 2000), in HEPES-buffered frog Ringer’s et al., 1997; Zheng et al., 1999), but it remained to be and then were prepared as they were for time-lapse record- determined how hair cells might lose their hair bun- ings. Images were acquired every 24 h starting at the end of dles and whether the same cells then reform a new the gentamicin treatment. Image stacks were obtained using transduction apparatus. two-photon excitation at 870 nm in a laser scanning confo- We set out to test the hypothesis that hair cells cal microscope (Periasamy et al., 1999). Stacked z-section images were acquired at 0.5 ␮m intervals starting above the could lose their apical components, survive in the level of the hair bundles and ending below the bottom of the epithelium, and then rebuild their hair bundle. We epithelium. Image stacks were taken from the same regions treated bullfrog saccules with a low dose of amino- in the saccules at 24 h intervals starting at the end of the glycoside antibiotics for 16–18 h and undertook long- 16–18 h gentamicin treatment. xz views of the epithelium term time-lapse and two-photon microscopic record- were reconstructed from 120–140 individual two-photon ings in order to follow the damage and recovery of the sections of the epithelium. hair cells. We also used scanning and transmission electron microscopy (SEM and TEM) as well as im- Immunocytochemistry munocytochemistry in determining how the damage and recovery processes proceeded. Thirty-six saccules, 12 each at 2, 4, and 7 days post- treatment, were placed in medium containing 1 ␮M ethidium homodimer, a marker of dead cells (Molecular MATERIALS AND METHODS Probes), for 30 min to test for cell death. To positively confirm the viability of surviving cells 2 days after the Organ Culture antibiotic treatment, two saccules were placed in medium that contained 1 ␮M Calcein-AM (Molecular Probes), Sixty-five bullfrog (Rana catesbiana) saccules were dis- which is cleaved by esterases and is thus a marker of live sected in HEPES-buffered frog Ringer’s (pH 7.25, 110 mM cells. NaCl, 2 mM KCl, 3 mM glucose, 10 mM HEPES) contain- The hair cell antibody, HCS-1, was generated by immu- ing 0.1 mM calcium chloride, then placed in 50 ␮g/mL nizing mice with sensory epithelia isolated from the utricles subtilisin (protease type VIII; Sigma, USA) in calcium-free of 1- to 21-day-old White Leghorn chicks. The monoclonal frog Ringer’s for 15 min. The otolithic membrane was then antibody HCS-1 binds specifically to the cell bodies of hair removed using a gentle flow of Ringer’s. The organs were cells in inner ear tissues from rat, mouse, chick, frog, and Hair Bundle Repair in the Bullfrog’s Saccule 83 shark (Gale et al., 2000; Finley et al., 1997). Specimens cells at all time points examined [Fig. 1(B), arrow]. were incubated in purified antibody (1:1000) in PBS with Damaged hair cells exhibited abnormal cell shapes, 5% normal goat serum and 0.1% Triton X-100 overnight at with processes extending up to 10 ␮m from the cell 4°C, followed by Cy3-conjugated goat antimouse IgG sec- body [Fig.