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.

A quantitative analysis of the nerve fibers in the VIIIth nerve of Belgian Waterslager canaries with a hereditary sensorineural hearing loss.

The number of auditory nerve fibers was determined for non-Belgian Waterslager canaries (non-BWS) and Belgian Waterslager canaries (BWS) that are affected by a sensorineural high frequency hearing loss and a 30% reduction in the number of auditory hair cells. Counts were obtained from semithin cross sections of the Durcupan-embedded auditory nerve at the level of the internal auditory meatus. In addition, the number of lagenar fibers was determined from cross sections near the apical end of the cochlear duct in order to separate them from the total number of auditory nerve fibers. The mean number of auditory nerve fibers was 6076 in non-BWS and 5363 in BWS canaries, representing a 12% reduction in BWS. This small reduction in the number of auditory nerve fibers, as compared to the larger reduction in hair cell number, might be explained by a predominant loss of abneural hair cells in BWS, since it has been shown for other species that a large proportion of abneural hair cells are devoid of afferent innervation. In addition, we observed that despite the prominent hair cell pathologies documented for BWS canaries, the mean diameter of auditory nerve fibers from non-BWS canaries (2.22+/-0.81 microm) did not differ from those of BWS canaries (2.21+/-0.96 microm).

Neither endocochlear potential nor tegmentum vasculosum are affected in hearing impaired belgian waterslager canaries.

We previously showed that the Belgian Waterslager canary strain is affected by a hereditary hearing loss that is associated with a reduced number of hair cells and hair cell pathologies in the basilar papilla. Since hair cell pathologies were also present in the sacculus, Weisleder et al. (1994) suggested that these birds are afflicted by Scheibe's like dysplasia, a cochleo-saccular defect. In mammals, cochleo-saccular defects are characterized primarily by the lack of an endocochlear potential and abnormalities in the Stria vascularis which only secondarily lead to hair cell loss (Steel and Bock, 1983; Steel, 1994; 1995). Here we report the endocochlear potential of six ears from three non-Belgian Waterslager canaries and three ears of two Belgian Waterslager canaries to decide if Waterslager canaries are affected by a cochleo-saccular or by a neuroepithelial defect. The mean endocochlear potential was 17.6+/-2. 5 mV in the non-Waterslager canaries and 20.3+/-0.6 mV in Waterslager canaries. In addition, and consistent with the presence of a normal endocochlear potential, light microscopy of the tegmentum vasculosum provided no evidence for pathology. These data show that Belgian Waterslager canaries are affected by a neuroepithelial rather than a cochleo-saccular inner ear defect.

Avian species differences in susceptibility to noise exposure.

Previous studies of hair cell regeneration and hearing recovery in birds after acoustic overstimulation have involved relatively few species. Studies of the effects of acoustic overexposure typically report high variability. Though it is impossible to tell, the data so far also suggest there may be considerable species differences in the degree of damage and the time course and extent of recovery. To examine this issue, we exposed four species of birds (quail, budgerigars, canaries, and zebra finches) to identical conditions of acoustic overstimulation and systematically analyzed changes in hearing sensitivity, basilar papilla morphology, and hair cell number. Quail and budgerigars showed the greatest susceptibility to threshold shift and hair cell loss after overstimulation with either pure tone or bandpass noise, while identical types of overstimulation in canaries and zebra finches resulted in much less of a threshold shift and a smaller, more diffuse hair cell loss. All four species showed some recovery of threshold sensitivity and hair cell number over time. Canary and zebra finch hearing and hair cell number recovered to within normal limits while quail and budgerigars continued to have an approximately 20 dB threshold shift and incomplete recovery of hair cell number. In a final experiment, birds were exposed to identical wide-band noise overstimulation under conditions of artificial middle ear ventilation. Hair cell loss was substantially increased in both budgerigars and canaries suggesting that middle ear air pressure regulation and correlated changes in middle ear transfer function are one factor influencing susceptibility to acoustic overstimulation in small birds.

Vestibular function in Belgian Waterslager canaries (Serinus canarius).

The purpose of this study was to measure vestibular function in Belgian Waterslager canaries using short latency vestibular evoked potentials (VsEPs) elicited by linear acceleration stimuli. Responses were recorded with vertex to mastoid leads using traditional signal averaging. Response thresholds, latencies, and amplitudes were quantified and compared to non-Waterslager controls. Cochlear and vestibular organs were also processed for scanning electron microscopy. Results indicated that vestibular response thresholds were slightly, but significantly, higher for Belgian Waterslager canaries and response amplitudes at 0 dBre: 1.0 g/ms were significantly reduced compared to non-Waterslagers. Response peak latencies were not significantly different. The most striking morphological finding was that the stereociliary bundles of Waterslager saccular hair cells showed no common orientation. Previous reports have also found significantly less hair cells in Waterslager saccules (Weisleder and Park, Hear. Res. 80 (1994) 64-70); however, the present study did not confirm this finding. The utricle and ampullae appeared normal. The present results indicate that vestibular neural function, as measured with VsEPs, is affected in Belgian Waterslager canaries. The results also suggest that one structural correlate of the functional loss is the disordered stereociliary bundles in the sacculus.

Effects of columella removal on inner ear morphology in the chick.

We are currently removing the single middle ear bone (columella) in the domestic chick to introduce chemical agents directly into the inner ear. Since we are interested in the effect of these agents on neural structures within the avian basilar papilla (BP), we are concerned about any subtle changes that might result from the surgical procedure of columella removal alone. The purpose of this study was to use light and transmission electron microscopy to analyze morphological changes in the inner ear after columella removal. Fifteen-day-old chicks underwent a unilateral, bilateral or a sham removal of the columella. After columella removal, the oval window was either plugged with Gelfoam or Kimwipe (standard accepted procedure to prevent possible perilymph leak) or left uncovered. After a 5-day survival period, morphological changes were observed in the tegmentum vasculosum (TV) of all ears receiving a columella removal as compared to unoperated ears. Further, ears with Gelfoam plugging the oval window also had damage to the hair cells and support cells of the basilar papilla. In contrast, there were no observable differences in either auditory afferent or efferent nerve terminals on hair cells in the BP from any ears that had the columella removed compared to those from unoperated ears. These results suggest that columella removal alone may produce morphological changes to the TV within 5 days of surgery but not to structures within the BP. On the other hand, columella removal with a Gelfoam plug results in damage not only to the TV but also to cells within the basilar papilla during this same survival time. Despite damage to other structures within the inner ear, cochlear efferent and afferent terminals on surviving hair cells were unaffected by columella removal with or without plugging.

Recovery of hearing and vocal behavior after hair-cell regeneration.

Postmitotic hair-cell regeneration in the inner ear of birds provides an opportunity to study the effect of renewed auditory input on auditory perception, vocal production, and vocal learning in a vertebrate. We used behavioral conditioning to test both perception and vocal production in a small Australian parrot, the budgerigar. Results show that both auditory perception and vocal production are disrupted when hair cells are damaged or lost but that these behaviors return to near normal over time. Precision in vocal production completely recovers well before recovery of full auditory function. These results may have particular relevance for understanding the relation between hearing loss and human speech production especially where there is consideration of an auditory prosthetic device. The present results show, at least for a bird, that even limited recovery of auditory input soon after deafening can support full recovery of vocal precision.

Recovery of noise-induced changes in the dark cells of the quail tegmentum vasculosum.

Morphologic changes in the tegmentum vasculosum (TV) of adult quail after high intensity sound exposure were studied. Quail were continuously exposed to 115 dB SPL, 1500 Hz pure tone in a sound field for 12 h and either sacrificed immediately (0 day), 1, 2, 3, 4, 6 or 10 days later. Serial sections through the basilar papilla at 100 micron intervals from base to apex were obtained for study with light microscopy and TEM. Significant morphologic changes were found within the TV of quail sacrificed on days 0-4. On a quantitative scale, the majority of recovery occurred within the first 24 h. After four days survival the tegmentum appeared nearly normal. This recovery correlates well with the temporal pattern of threshold shift recovery. These results demonstrate a temporal correlation between ultrastructural changes in the TV and functional recovery of hearing after intense sound exposure. A potential etiologic role of the TV in avian temporary threshold shift is suggested.

TEM analysis of neural terminals on autoradiographically identified regenerated hair cells.

Regenerated tall and short hair cells identified by autoradiography ([3H]thymidine) were analyzed for their neural contacts using transmission electron microscopy. Ears from mature Coturnix quail (N = 5) exposed to pure tone overstimulation (1500 Hz, 115 dB, 12 h) and treated with [3H] thymidine for 10 days were fixed, embedded, sectioned serially in 100 mu intervals and prepared for autoradiography. At fifty percent length along the papilla, alternating semi-thick (1 micron) and thin (70 nm) sections were taken at 50 microns intervals. Semi-thick sections were analyzed at the light microscope level for autoradiographic labeling of [3H]thymidine over the hair cell nucleus. When an autoradiographically labelled hair cell was identified the corresponding serial thin sections were analyzed in the transmission electron microscope. Seven autoradiographically labeled hair cells in semi-thin sections were positively identified in immediately adjacent thin serial sections. Labeled hair cells were morphologically similar to adjacent cells with no label and generally appeared to receive similar innervation. Regenerated short hair cells showed large chalice shaped, efferent terminals, intermediate hair cells received both afferent and efferent innervation and tall hair cells were contacted by two to three afferent terminals with synaptic specializations. These results provide conclusive evidence of both efferent and afferent synaptic contacts on newly regenerated hair cells of all types 10 days following acoustic trauma.

Changes in the acoustic nerve after hair cell regeneration.

Hair cells of the avian inner ear have been shown to regenerate following acoustic or ototoxic insult. The consequences of this regeneration on the acoustic nerve have yet to be defined. The purpose of the present study was to use TEM analysis following cochlear damage and hair cell regeneration to describe afferent and efferent neural terminals on hair cells in the newly repopulated sensory epithelium. Following acoustic overstimulation (12 h, 115 dB SPL, 1500 Hz) adult quail were sacrificed immediately (0 day), or at 2, 12, or 24 weeks. Serial thin sections were taken from the embedded papilla in a plane tangential to the basilar membrane in the area consistent with regenerative activity. Immediately following noise exposure very few hair cells could be seen within the epithelia; afferent terminals on remaining cells appeared normal. Two weeks later afferent terminals showed signs of degeneration; efferent terminals were rarely seen on tall hair cells but remained relatively normal on short hair cells. Three to six months later afferent terminals had regained a more normal appearance but were less numerous on tall hair cells; some return of efferent-like terminals was seen often contacting two tall hair cells. Large normal appearing, efferent terminals remained on short hair cells. These results suggest that regenerated hair cells are likely to receive neural innervation. It would appear that some degeneration of afferent terminals takes place prior to final innervation of new hair cells.

Hair cell regeneration in the chicken cochlea following aminoglycoside toxicity.

Hair cell loss in the avian cochlea partially recovers following both acoustic trauma and aminoglycoside intoxication. DNA labeling with tritiated thymidine has shown that the restoration of cell number following acoustic trauma results from the production of new hair cells by mitotic division. The purpose of the present study was to determine if mitosis also contributes to the recovery of hair cell number which occurs following aminoglycoside intoxication. Chickens received daily injections of either gentamicin sulfate or distilled water for 10 consecutive days. During the latter 7 days of this period, all birds were also injected with [3H]thymidine. Following postinjection survival periods of 3 or 6 days, one papilla from each bird was processed for autoradiography and the other for scanning electron microscopy (SEM). Incorporation of [3H]thymidine was seen over hair cells and support cells in experimental papillae in regions of hair cell loss. No labeling was seen outside of damaged regions or in the papillae of control birds. SEM showed that damaged regions in experimental birds contained cells similar in appearance to developing auditory hair cells in avian embryos. These results show that the restoration of hair cell number following aminoglycoside toxicity results from the production of new cells by mitosis.

Issues in neural plasticity as related to cochlear implants in children.

There is compelling evidence across species for a changing place code during development. This change in frequency organization may provide a mechanism for all elements within the central auditory pathways to receive the necessary stimulation to promote normal growth and development. We must take these normal developmental processes into consideration when deciding on the appropriate stimulation, training, and success procedures in cochlear implants in children.

Hair cell regeneration in senescent quail.

Hair cell regeneration was studied following exposure to an intense pure tone stimulus in young adult and senescent Coturnix quail. Three, 3-month old and four, 3-year old quail were continuously exposed to a 1500 Hz pure tone at 115 dB SPL for 12 h. Four quail were not noise exposed and were used as age-matched controls. Control and experimental birds received injections of [3H]thymidine daily for 10 days after noise exposure. Ten days after noise exposure birds were killed and their cochleae embedded, sectioned serially and processed through standard methods of autoradiography. Hair cell counts showed a discreet area of hair cell loss for both age groups in the proximal half of the papilla. Incorporation of [3H]thymidine was clearly seen over the nuclei of hair cells and support cells in the region of hair cell loss in both age groups. Incorporation of [3H]thymidine was also seen over the nuclei of hair cells and support cells in a very small area in two of the non-exposed control birds. These results demonstrate that the potential for hair cell regeneration is maintained throughout life in Coturnix quail. Further, they suggest that there may be some very low level of hair cell production in the normal adult quail ear which is activated in the absence of massive trauma.

Ganglion cell loss continues during hair cell regeneration.

Hair cells and ganglion cells were counted in young adult quail (Coturnix coturnix) after acoustic trauma at 10, 30, 60 and 90 day survival times. Following sacrifice the basilar papillae, along with the ganglia, were fixed, embedded in plastic and sectioned serially at 100 mu intervals from basal to apical tip. Hair cells and ganglion cells were counted from 3 mu thick sections at each interval. Hair cells were designated as tall or short within the area 30-70% of length from basal tip of the papilla. Both tall and short hair cells were significantly reduced in number 10 days following trauma. Tall hair cells recovered to within 96% of normal after 60-90 days. Short hair cells recovered but to a lesser extent. Ganglion cell loss did not begin until 30 days after trauma and continued without recovery 90 days after trauma. A good correlation was found for position of both types of hair cell loss and position of ganglion cell loss. These results suggest that the initial loss of hair cells, both tall and short, results in retrograde degeneration of neural fibers and ganglion cells.

Ganglion cell and hair cell loss in Coturnix quail associated with aging.

Hair cells and ganglion cells were examined in young adult (3 month old) and senescent (3 to 6 year old) quail (Coturnix coturnix). Following sacrifice the basilar papillae, along with the ganglia, were fixed, embedded in plastic and sectioned serially at 100 micron intervals from basal to apical tip. Hair cells and ganglion cells were counted from three micron thick sections at each interval. Hair cell number remained constant between age groups (less than 10% loss even in the oldest group). Ganglion cell number, on the other hand, was considerably reduced in the senescent birds (20-60% loss). These results are similar to quantitative results in senescent mammals and suggest that ganglion cell loss may be generalized response to aging.

Hair cell regeneration after acoustic trauma in adult Coturnix quail.

Recovery of hair cells was studied at various times after acoustic trauma in adult quail. An initial loss of hair cells recovered to within 5 percent of the original number of cells. Tritium-labeled thymidine was injected after this acoustic trauma to determine if mitosis played a role in recovery of hair cells. Within 10 days of acoustic trauma, incorporation of [3H]thymidine was seen over the nuclei of hair cells and supporting cells in the region of initial hair cell loss. Thus, hair cell regeneration can occur after embryonic terminal mitosis.

The effects of reduced cerebrovascular circulation on the auditory brain stem response (ABR).

The effect of decreased anterior (carotid) bloodflow on the auditory brain stem response (ABR) was studied in a group of middle-aged males with angiographically confirmed decreased carotid circulation who were candidates for internal carotid end-arterectomy. Individual and mean latencies from the experimental group were compared to latencies of a control group matched for age, sex, and hearing loss. Mean absolute latency of wave V was significantly longer in the group with decreased anterior circulation when compared to the matched control group. These findings are similar to those from studies examining decreased posterior (vertebrobasilar) circulation. Both the matched control and experimental group were also compared to clinic norms and judged for abnormality. None of the control group ABRs were judged abnormal, after correction for hearing loss, while 29% of the ABRs were judged abnormal from the group with decreased anterior circulation. Postoperative testing was performed 1 week after surgery to determine any immediate effects of revascularization of the ABR. No changes in absolute or interpeak latencies were observed for either ear between pre- and postoperative testing. It is concluded that patients with confirmed decreased cerebrovascular circulation may show prolongation of absolute latency of wave V on ABR testing. Revascularization surgery in these patients appears to have no affect on their ABR within 1 week of surgery.

Ontogenetic changes in the position of hair cell loss after acoustic overstimulation in avian basilar papilla.

High intensity sound was used to produce localized hair cell damage within the basilar papilla of chicks at three different ages: embryonic day 20, post-hatch day 10 and post-hatch day 30. At each age separate groups of animals were exposed to broadband white noise or pure tones at 500, 1500 or 3000 Hz for 12 h at 125 dB SPL. Chicks were killed 10 days later. Their basilar papillae were then fixed, dissected free, osmicated, embedded in Epon, sectioned serially and stained. Hair cells were counted at 100 micron intervals throughout the length of the papilla. There was a systematic developmental shift in the position of damage produced by each of the acoustic stimuli. Broadband white noise produced damage only in the basal one half of the cochlea in the embryonic animals while at later ages it produced damage throughout the length of the papilla. Exposure with each of the pure tones produced a discrete area of hair cell loss. However, with each frequency the region of damage shifted apically as a function of the age of the animal at the time of sound exposure. These results suggest that the frequency representation along the basilar papilla is not fixed, but changes during the development of hearing.

Differential susceptibility of avian hair cells to acoustic trauma.

Five groups of 10-day-old chicks were continuously exposed to either 500 or 1500 Hz pure tone at 125 dB for 4 or 12 h and killed 10 days later. The basilar papillae were fixed, embedded in plastic, sectioned, and hair cells were counted according to type: tall or short. Short hair cells were found to be more susceptible to acoustic overstimulation than tall hair cells. Further, the position of maximum short hair cell loss varied along the length of the basilar papilla as a function of the exposure frequency while the position of tall hair cell loss did not. Similarities between morphological response of short hair cells in avians after acoustic trauma and outer hair cells in mammals are discussed.