Expression and localization of prestin and the sugar transporter GLUT-5 during development of electromotility in cochlear outer hair cells.

Electromotility, i.e., the ability of cochlear outer hair cells (OHCs) to contract and elongate at acoustic frequencies, is presumed to depend on the voltage-driven conformational changes of "motor" proteins present in the OHC lateral plasma membrane. Recently, two membrane proteins have been proposed as candidates for the OHC motor. A sugar transporter, GLUT-5, was proposed based on its localization in the OHCs and on the observation that sugar transport alters the voltage sensitivity of the OHC motor mechanism. Another candidate, "prestin," was identified from a subtracted OHC cDNA library and shown to impart voltage-driven shape changes to transfected cultured cells. We used antibodies specific for these two proteins to show that they are highly expressed in the lateral membrane of OHCs. We also compared the postnatal expression patterns of these proteins with the development of electromotility in OHCs of the apical turn of the rat organ of Corti. The patch-clamp recording of transient charge movement associated with electromotility indicates that half of the maximal expression of the motor protein occurs at postnatal day 9. Prestin incorporation in the plasma membrane begins from postnatal day 0 and increases progressively in a time course coinciding with that of electromotility. GLUT-5 is not incorporated into the lateral plasma membrane of apical OHCs until postnatal day 15. Our results suggest that, although GLUT-5 may be involved in the control of electromotility, prestin is likely to be a fundamental component of the OHC membrane motor mechanism.

A gene upregulated in the acoustically damaged chick basilar papilla encodes a novel WD40 repeat protein.

The chick WDR1 gene is expressed at higher levels in the chick basilar papilla after acoustic overstimulation. The 3.3-kb WDR1 cDNA encodes a novel 67-kDa protein containing nine WD40 repeats, motifs that mediate protein-protein interactions. The predicted WDR1 protein has high sequence identity to WD40-repeat proteins in budding yeast (Saccharomyces cerevisiae), two slime molds (Dictyostelium discoideum and Physarum polycephalum), and the roundworm (Caenorhabditis elegans). The yeast and P. polycephalum proteins bind actin, suggesting that the novel chick protein may be an actin-binding protein. Sequence database comparisons identified mouse and human cDNAs with high sequence identity to the chick WDR1 cDNA. The mouse Wdr1 and human WDR1 proteins showed 95% sequence identity to each other and 86% identity to the chick WDR1 protein. Northern blot analysis of total RNA from the chick basilar papilla after noise trauma revealed increased levels of a 3.1-kb transcript in the lesioned area. The WDR1 gene was mapped to human chromosome 4, between 22 and 24 cM from the telomere of 4p.

Morphometric changes in the chick nucleus magnocellularis following acoustic overstimulation.

The present investigation considered the effects of cochlear damage caused by exposure to intense sound on the nucleus magnocellularis of the chick. Neonatal chicks exposed to intense sound were separated into four groups with post-exposure recovery durations of 0, 15, 27, and 43 days. Four age-matched, non-exposed control groups were also formed. At each recovery interval, the control and exposed birds were sacrificed and their brains prepared for paraffin embedding. The brain stem region containing the nucleus magnocellularis (NM) was serially sectioned in the coronal plane. All sections containing NM cells were identified and then coded in terms of their percentile distance from the most caudolateral section. Sections along the nucleus at the 15th, 30th, 50th, 65th, 80th, and 95th percentile positions were selected for evaluation, and the cross-sectional areas of individual NM cells in these sections were then measured. Cell areas were corrected for the bias introduced by eccentricity of the nucleus. The number of NM cells per 1,000 microm2 was also calculated at the 50th and 65th percentile positions. These procedures were repeated for the age-matched, non-exposed control animals. The cross-sectional cell area in exposed animals, immediately after the exposure, was reduced significantly at all positions, but returned to near normal by 43 days of recovery. However, the coronal area of NM in the sections at the 50th and 65th percentile position, as well as NM cell density, were unaffected by the exposure at all recovery intervals. The observation of structural recovery in NM cells at 43 days post-exposure was remarkable because it occurred at least 4 weeks after complete functional restoration of single-cell activity in the NM. The shrinkage in NM cell size throughout the nucleus may be due to a general reduction in spontaneous activity in the cochlear nerve fibers caused by the acoustic injury to the chick basilar papilla.

Further evidence for supporting cell conversion in the damaged avian basilar papilla.

Two lines of evidence suggested that a process other than supporting cell divisions may give rise to new hair cells in the bird inner ear injured by either noise or ototoxic drugs. This process, supporting cell conversion, occurs when non-dividing supporting cells transdifferentiate into hair cells. First, noise-exposed chicks received zero, one or two daily i.p. injections of cytosine arabinoside (a DNA synthesis blocker), as well as two daily intraperitoneal injections of bromodeoxyuridine, for four days. Following sacrifice, the papillae were processed for bromodeoxyuridine immunocytochemistry. All the ears demonstrated dividing cells, but increasing the number of cytosine arabinoside injections decreased the number of labeled cells. Indeed, two cytosine arabinoside injections per day nearly completely blocked supporting cell divisions in the short hair cell region within the sound-induced lesion. This suggested that unpaired, immature cells observed in a similar region with scanning electron microscopy, despite the presence of cytosine arabinoside, may have been products of supporting cell conversion. In the second experiment, birds were treated with gentamicin for three days. Upon sacrifice at 6 days post-treatment, papillae were processed for light and transmission electron microscopy. Several unusual cells were observed with phenotypic features of both hair cells and supporting cells. The peculiar cells may be in a transition from the supporting cell phenotype to that of a hair cell.

Identification of genes expressed after noise exposure in the chick basilar papilla.

We used differential display of mRNA, a method based on reverse transcriptase-PCR, to identify genes whose expression increases in response to acoustic trauma in the chick basilar papilla. Identifying these genes would provide insight into processes involved in repair of the damaged epithelium or in hair cell regeneration. We compared mRNA from the basilar papilla of normal chicks, from chicks exposed to an octave band noise (center frequency: 1.5 kHz) presented at 118 dB for 6 h, and from chicks exposed to noise and allowed to recover for 2 days. Thus far, we have identified 70 bands that appear to be differentially displayed on DNA sequencing gels; approximately 40 of these bands have been subcloned and sequenced. DNA sequences were compared with sequences in the GenBank database to identify genes with significant (70-85%) sequence identity to known genes. Chick cDNAs identified included: the parathyroid hormone-related protein, an immediate early gene; the delta-subunit of the neuronal-specific Ca2+/calmodulin-regulated protein kinase II; and the GTP-binding protein CDC42, a member of the ras superfamily of G proteins. A fourth cDNA had 84% sequence identity to an uncharacterized human cDNA (expressed sequence tag), indicating that this is a novel gene. Slot-blot hybridization analysis of these cDNAs probed with labeled DNA generated from mRNA from each experimental group indicated higher levels of mRNA for each of these four genes after noise exposure. These results indicate the potential involvement of both Ca2+/calmodulin-mediated signaling and GTPase cascades in the response to noise damage and during hair cell regeneration in the chick basilar papilla.

Tectorial membrane repair in the quail following multiple exposures to intense sound.

Adult quail were exposed one, two or three times to an intense octave band noise. Birds were sacrificed 0 or 14 days after the last exposure and their ears processed for scanning electron microscopy (SEM). SEM observations demonstrated a patch lesion in every ear at 0 and 14 days after exposure. In the single-exposed ear immediately after noise, the lesion lacked any tectorial membrane (TM). At 14 days of recovery, the site of injury was covered by a partially regenerated TM (referred to as the honeycomb). However, the patterns of TM damage and repair varied among the twice- and thrice-exposed papillae. At 0 days after exposure, the multiple-exposed ear displayed a lesion with two segments. In one portion the TM was missing, and in the other portion the TM was present as one or two honeycomb layers. Fourteen days later, a new honeycomb was observed underneath the remaining honeycomb. These results suggest that the quail is able to repeatedly repair its TM following multiple noise exposures.

New hair cells arise from supporting cell conversion in the acoustically damaged chick inner ear.

Supporting cell mitosis contributes significantly to hair cell regeneration in the acoustically damaged bird inner ear. Yet there may be another mechanism of hair cell replacement: supporting cell conversion. This study used cytosine arabinoside (Ara-C), an inhibitor of DNA synthesis, to better determine whether supporting cells could transdifferentiate into hair cells without cell division. Chicks received Ara-C injections after acoustic overstimulation. Scanning microscopic studies of the basilar papillae revealed several unpaired, immature hair cells. To ensure Ara-C's blockage of DNA synthesis, one group of birds received both Ara-C and bromodeoxyuridine (BrdU), while another group had BrdU only. Immunocytochemical analysis of Ara-C/BrdU and BrdU papillae indicated zero and 16 dividing cells, respectively. This difference confirmed that Ara-C blocked DNA synthesis, arresting supporting cell mitosis. These data strongly suggest that supporting cell can convert into hair cells.

Comparative analysis of patch lesions in the chick inner ear following acoustic trauma: optical versus scanning electron microscopy.

Neonatal chicks were exposed to an octave band noise with a center frequency of 1.5 kHz at 116 dB SPL for 4 hours. Seven days following overstimulation, the birds were sacrificed. Their basilar papillae were removed, fixed in 4% paraformaldehyde, and processed in two steps. First, the ears were immunostained with a supernatant of mouse anti-tectorial membrane antibodies, followed by a diaminobenzidine process. Examinations of the papillae under an optical stereo microscope revealed a patch site with a partially regenerated tectorial membrane (referred to as the honeycomb). After the optical studies, the same ears were post-fixed in 1% osmium tetroxide, dehydrated in ethanol, and processed for scanning electron microscopy (SEM). SEM examinations demonstrated a honeycomb-covered patch lesion in the papilla. Patch lesion perimeters were traced from both the optical and SEM images, and patch areas were calculated. Also, papilla height was measured at the midpoint of the inner ear in both groups. These calculations showed that the patch area and papilla height had shrunk by approximately 37% and 33%, respectively, following the SEM methodology. The decrease in these dimensions may be attributed to several steps required for the SEM specimen preparation, such as critical point drying.

Tectorial membrane regeneration in acoustically damaged birds: an immunocytochemical technique.

A novel immunocytochemical method was used to determine whether the sound-damaged adult quail ear can repair its tectorial membrane (TM) and to compare the repair in quail to that in chicks. Birds were exposed to an octave band noise with a center frequency of 1.5 kHz at 116 dB SPL for 4 h. The chicks were grouped based on recovery duration (0 and 7 days), while the quail were divided into 0-, 7-, and 14-day recovered groups. At the end of the recovery period, the animals were sacrificed, and their basilar papillae labeled with a TM-specific monoclonal primary antibody solution followed by a diaminobenzidine process. Examinations under a stereoscope revealed that a patch lesion devoid of TM was located on all 0-day recovered papillae. Seven days later, a honeycomb-patterned layer was observed covering the lesion. In 14-day recovered quail ears, the honeycomb layer appeared similar to that seen at 7 days post-exposure. These observations indicated that both chicks and quail were able to repair their TM within 7 days following exposure to intense sound.

Hair cell replacement in the avian inner ear following two exposures to intense sound.

The present study is concerned with the degree to which the avian cochlea retains the capacity to regenerate hair cells following repeated exposures to intense sound. Two groups of chicks were exposed once to an intense pure tone for 48 h at either 2 or 16 days of age. In a third group, they were exposed to the same stimulus at both ages. Structural alterations of the auditory epithelium were assessed both qualitatively and quantitatively at 0, 12 or 26 days following the single exposure at 2 days of age, and at 0 or 12 days following the single or second exposure at 16 days of age. The numbers of hair cells lost in the twice-exposed birds and those exposed once at 2 days of age were approximately 24 and 36%, respectively, and were not significantly different. Interestingly, the single exposure at 16 days of age caused greater hair cell loss (61%). Twelve days after overstimulation, the hair cell population in all experimental groups returned to near normal levels due to the emergence of new hair cells. This observation of hair cell replacement extends the early findings that birds are able to repair their acoustically damaged ears after either a single or repeated overexposure.

Cell cycle of transdifferentiating supporting cells in the basilar papilla.

Mitosis of supporting cells has been shown to contribute to the cellular repopulation of the basilar papilla after acoustic trauma. In the present work we report data obtained with light and transmission electron microscopy after acoustic trauma in chicks. We report changes that occur in cell shape, surface morphology, intercellular junctions, nuclear shape and location, and cytoplasmic organization of supporting cells after trauma. The findings strongly suggest that supporting cells transdifferentiate and that the proliferative pattern is similar to interkinetic nuclear migration, as previously shown in the developing neural tube and basilar papilla. S-phase nuclei were positioned adjacent to the basement membrane, suggesting that interaction with the extracellular matrix may occur during the cell cycle. Supporting cells divided with the long axis of the spindle parallel to the reticular lamina and displayed no signs of intercellular communication during mitosis. This suggested to us that the fate of the progeny cells is determined prior to mitosis and that the progeny may be of identical phenotypic fate. Dividing cells had a smooth apical surface. The smooth surface may provide a marker to help identify dividing cells with scanning electron microscope analysis.

Recovery of auditory function and structure in the chick after two intense pure tone exposures.

Groups of neonatal chicks were examined in three experimental conditions that differed in the age and number of times they were exposed to a pure tone of 0.9 kHz at 120 dB SPL for 48 h. Several animals were exposed once at 2 or 16 days of age, while others were subjected twice to the above stimulus, first at 2 days and then at 16 days. Evoked potential measures of threshold shift, obtained at 0, 12 or 26 days post-exposure, were used to determine the degree of hearing loss and recovery. The average threshold loss in the mid-range frequencies was about 57 dB at 0 days for all three conditions. This level was reduced to about 15 dB in all three groups at 12 days of recovery, while in birds exposed once at 2 days, but allowed 26 days to recover, the post-exposure thresholds returned to pre-exposure levels. Scanning electron microscopic analysis of cochlear structure was conducted in groups of similarly exposed and recovered animals. Twelve days post-exposure, the structural analysis revealed regeneration of a single honeycomb-like tectorial membrane layer in both the once and twice-exposed cochleae. However, damage to, and repair of, the tectorial membrane after the second exposure revealed the production of a second honeycomb layer in about half the animals examined. The results indicated that chicks retain the capacity to repair receptor epithelium damage and recover considerably from hearing loss after multiple exposures to intense sound.

Growth of evoked potential amplitude in neonatal chicks exposed to intense sound.

The recovery of auditory function at selected intervals following exposure to a 0.9 kHz tone for 48 h at 120 dB SPL is described in neonatal chicks. Evoked potentials recorded from the nucleus magnocellularis were used to measure threshold sensitivity and peak-to-peak response amplitude as a function of stimulus intensity. The relation between evoked-response amplitude and stimulus intensity was nearly linear in control animals. However, at 10 days post exposure, the evoked response in mid-range frequencies showed a severe threshold shift and an abnormally rapid growth in amplitude. At 3 days post exposure, the rate of growth was nearly identical to that measured in control animals and threshold sensitivity showed considerable recovery. Current theories of amplitude-intensity growth and studies of basilar papilla damage and repair following intense sound exposure were applied in the analysis of these results.

Threshold shift, hair cell loss, and hair bundle stiffness following exposure to 120 and 125 dB pure tones in the neonatal chick.

One-day old chicks were exposed to one of two pure tone stimuli (0.9 kHz at 120 or 125 dB SPL) for 48 h. Three major results arose from a variety of tests that assessed the structural and functional consequences of the exposure on the peripheral auditory system at either 0 days or 12 to 15 days recovery. First, brainstem response data showed that the 120 and 125 dB groups had maximum evoked potential threshold shifts of 57 and 71 dB immediately after removal from the sound. Fifteen days post-exposure, the thresholds in the 120 dB group returned to near-normal levels, while in the 125 dB group, recovery was within 19 dB of control thresholds. Second, scanning electron microscopic measurements of hair cell density within the lesion showed that at 0 days recovery, the 120 and 125 dB groups had a 30% and 59% short hair cell loss, respectively, but by 15 days no differences could be identified between the exposed and control animals, regardless of exposure level. Finally, at 0 days of recovery, micromechanical stimulation data did not reveal any significant difference in stiffness between the control and surviving hair cells in the lesion area. Although the more intense exposure induced greater structural and functional damage in the chick cochlea, the birds retained or even enhanced their ability to replace lost hair cells and had partial hearing recovery by 15 days post-exposure.

The structural and functional aspects of hair cell regeneration in the chick as a result of exposure to intense sound.

This paper summarizes the structural and functional damage caused by intense sound exposure in neonatal chicks. Scanning electron microscopy has been used to follow the structural changes to the papilla and their subsequent repair. Pure-tone exposures produced a localized lesion consisting of tectorial membrane destruction, changes in surface organization of the papilla, and hair cell loss. The papilla underwent significant repair following the exposure and new hair cells could be identified on the sensory surface after 4 days of recovery. In addition, various evoked-potential methods provided an objective assessment of auditory function and demonstrated that the peripheral ear was severely impaired by overstimulation. Auditory function returned to near normal levels within 3 days postexposure. The inescapable conclusion from these observations was that hair cell regeneration had little to do with the functional recovery observed during the first 3 days. Tectorial membrane regeneration and the restoration of cochlear micromechanics were combined to form a hypothesis to account for the restoration of auditory function.

Biochemical analysis of solubilized angiotensin II receptors from murine neuroblastoma N1E-115 cells by covalent cross-linking and affinity purification.

Angiotensin II (Ang-II) receptors were solubilized from differentiated N1E-115 neuroblastoma cell membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), whereas other detergents, such as digitonin, sodium cholate, and Triton X-100, were much less effective. Binding of 125I-Ang-II or the antagonist 125I-Sar1,Ile8-Ang-II to 1% CHAPS-solubilized membranes was saturable and of high affinity. Moreover, these solubilized receptors retained the pharmacological specificity characteristic of particulate receptors. Covalent cross-linking of 125I-Ang-II to either particulate or solubilized membrane fractions, with the homobifunctional cross-linker disuccinimidyl suberate, followed by size exclusion chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, resulted in the identification of the same two distinct 125I-Ang-II binding entities, with approximate molecular masses of 111 kDa and 68 kDa. The estimated molecular weights of the Ang-II binding sites in differentiated N1E-115 cells are in good agreement with the molecular weights obtained previously from solubilized rat brain membranes, suggesting that the N1E-115 Ang-II receptors are similar to those present in the brain. Finally, solubilized N1E-115 membranes could be purified by Ang-II affinity chromatography, resulting in only a single protein (66 kDa), which retained its ability to specifically bind 125I-Ang-II.